Science topic

Antibodies - Science topic

An antibody, also known as an immunoglobulin, is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen.
Questions related to Antibodies
  • asked a question related to Antibodies
Question
1 answer
I'm looking for a Crhr2 antibody suitable for immunofluorescence in mouse brain slices. I've tried ab236982 from Abcam, but it didn't work well for me.
Does anyone have any other recommendations?
Thanks!
Relevant answer
Answer
Hi, have you found the antibody?
  • asked a question related to Antibodies
Question
1 answer
I have made TL reagent by mixing antibody in 1X PBS containing 0.25% sucrose for striping similarly the CL which is anti species of TL and prepared using the above composition. While running the LFA, I can see both TL and CL increases linearly with my analyte concentration which was strange.
I have found out with my investigation that my test line antibody move during the running of the lateral flow and the TL antibody gets captured at the CL and forms the sandwich with the analyte there and hence I see the linear correlation with the analyte.
I need to know how can I immobilize my antibody firmly to the nitrocellulose membrane, such that it does not move and interfere with CL antibody.
Thanks,
Yogita
Relevant answer
Answer
These comments may help
1-Increase your sucrose concentration up to 4%
2-in addition to antibody add 1-2mg/ml BSA to PBS
3-add about 50-60 microliter alcohol/ml
  • asked a question related to Antibodies
Question
2 answers
Hi,
After coating a 48-well plate with a monocyte-specific antibody, then adding monocytes, it is impossible to harvest the cells (i.e. with vigorous pipetting, trypsin, or EDTA).
If there are any tips to harvest the cells in this scenario I'd be grateful to hear them.
Thanks very much,
Adam
Relevant answer
Answer
Thanks so much for the suggestion Hameer Khan Khaskheli
  • asked a question related to Antibodies
Question
1 answer
Does anybody know of any functional anti-CD89 antibody or other reagent to block human IgA from binding to CD89 on cells? Thank you.
Relevant answer
Answer
Dear Dr. Shi-Hsia Hwa
You could consider three approaches.
1. Use of monoclonal antibodies that specifically target the CD89 receptor. The antibodies can bind to CD89 and block its interaction with IgA, effectively neutralizing its activity.
2. Small molecules and peptides that can disrupt the CD89-IgA interaction can be designed to fit into the binding site of the receptor or the antibody, preventing them from coming together.
3. Another strategy would be to involve the use of decoy proteins that mimic the natural ligands of CD89 without triggering an immune response. These decoys can compete with IgA for binding to CD89, thereby reducing the effective interaction between the receptor and its natural ligand.
As far as antibodies are concerned, there are commercially available antibodies.
1. Mouse anti-human CD89 antibody clone MIP8a, which is known to recognize an epitope within the EC1 domain of human CD89, bound to ‘human EC1-CD89’ and not to ‘human EC2-CD89.'
2. Mouse anti-human CD89 antibody clone A59, which recognizes an epitope within the EC2 domain of human CD89, bound to ‘human EC2-CD89’ and not to ‘human EC1-CD89’.
3. Mouse anti-human CD89 antibody clone A3, which recognizes an epitope depending on parts from both EC1 and EC2 domains of human CD89, bound to ‘human EC2-CD89’ but not to ‘human EC1-CD89’.
For more information, you may want to refer to the articles provided below.
Additionally, an invention in the link below discloses an antibody that can bind an extra-cellular part of human CD89 (human FcaRI) on human CD89 expressing cells that prevents binding of human IgA to human CD89 when the antibody is bound to said cells and that induces less cell death in said human CD89 expressing cells when compared to the antibody MIP8a.
Regards,
Malcolm Nobre
  • asked a question related to Antibodies
Question
3 answers
Hi everyone,
I am currently facing a challenge in detecting phosphorylated FLT3 at the expected molecular weights of ~130 kDa (non-glycosylated form) and ~160 kDa (glycosylated mature form) in my Western blot experiments. Interestingly, I consistently observe a band at approximately 50 kDa, which is noted in the datasheet for the antibody.
Here are the specifics of my protocol:
  • Primary Antibody: Phospho-FLT3 (Tyr591) Antibody #3461 (Cell Signaling Technology).
  • Antibody Dilution: 1:250, which is four times more concentrated than the recommended 1:1000 dilution.
  • SDS-PAGE: I have employed both 8% and 12% gels.
  • Electrophoresis Conditions: 40 minutes at 200V, 0.24 AMPS.
  • Transfer Conditions: PVDF membrane activated and transferred for 2 hours at 100V, 0.35 AMPS.
  • Detection: SuperSignal™ West Femto Maximum Sensitivity Substrate.
Despite optimizing various conditions, I have not been able to detect phosphorylated FLT3 at the anticipated higher molecular weight ranges. The lower band at ~50 kDa is consistently present. I am considering whether this could be related to the antibody dilution, electrophoresis, or transfer conditions.
Has anyone encountered similar issues or could provide insights into potential adjustments to enhance the detection of the 130/160 kDa bands? Any recommendations on troubleshooting this would be highly appreciated.
Thank you for your time and expertise.
Relevant answer
Answer
I would suggest to try blocking with BSA for detecting phosphorylated proteins specifically because of the presence of phosphatases in milk.
  • asked a question related to Antibodies
Question
2 answers
After being modified with DBCO-PEG4-NHS and subsequently labeled with a secondary antibody (targeting the Fc region), the antibody was found to be unable to bind to antigen-expressing cells. What could be the possible reasons for this
Relevant answer
Answer
Actually; Every antibody is specific for the specific antigen, Any antibody which is for specific antigen that will no work or bind with other antigen, because light chain of antibody have specific site for antigen and when we modify antibody it will lose its specific binding site , so ,you may use alternative antibodies or may there an error with the procedure of doing and change the experimental condition for working
  • asked a question related to Antibodies
Question
1 answer
Starting some new cell line RNAseq experiments and looking for recommendations on capture antibodies against CAIX and EpCAM. Preferably mouse host and monoclonal. Thank you
Relevant answer
Answer
Creative Biolabs has recombinant mouse mAbs that might be of interest to you:
  • asked a question related to Antibodies
Question
4 answers
Hello everyone, I am trying to visualise ABCA1 protein (which is approximately 220-240 KD) by western blot from my experimental in-vitro cell samples. To extract proteins, I use either: NucleoSpin RNA/Protein extraction kit (Machery Nagel), or traditional cell lysis buffer or RIPA buffer. I can clearly see strong and clear bands for the house-keeping gene (actin), but the ABCA1 seems either, very faint, smear like or blurry. I am using ABCAM ABCA1 antibody, and loading the appropriate protein quantity as per the antibody manufacturer instructions. I am using either 1.5 or 1.0mm gel and bio-rad tetra system to do the run. Could anyone suggest how can i improve the abca1 band visualization ?. Thanks in advance.
Relevant answer
Answer
Samrat Das since your protein of interest is around 220-240 KDa, I suggest the following steps.
1. Use a low percentage gel about 7.5%
2. Optimize the transfer conditions. It's better to use wet transfer to visualize high molecular weight proteins. If you're uing the semi-dry method, transfer at 20V for 1 hour.
3. Increase the protein concentration.
  • asked a question related to Antibodies
Question
5 answers
I'm having trouble with my western blot. I'm using an antibody called L1CAM. It has appeared in previous blots I've ran but recently it doesn't show up at all in the last two I've done. I've made fresh antibody each time, the antibody hasn't expired, and the transfer was successful. I don't know why its not showing up. Below I attached an image of my blot.
Relevant answer
Answer
Hi Mike!!
Even though the antibody hasn't expired, it's possible that repeated freeze-thaw cycles have degraded it, which can affect its performance. If possible, try using a fresh vial or a different lot. You could also test the blot with an internal control antibody like GAPDH or another reliable marker on the same blot. If the control antibody works, it's a strong indication that your L1CAM antibody may be degraded.
  • asked a question related to Antibodies
Question
1 answer
Does anyone have a good antibody for Fsp1 (S100A4) that can be used for IHC on frozen sections (mouse tissue)? Thanks so much!
Relevant answer
Answer
Not a recommendation of quality, but you might want to look at those pAbs offered by both Biorbyt (https://www.biorbyt.com/s100a4-antibody-orb6910.html) and antibodies-online (https://www.antibodies-online.com/antibody/703706/anti-S100+Calcium+Binding+Protein+A4+S100A4+AA+15-101+antibody/)
  • asked a question related to Antibodies
Question
1 answer
As part of my work with antisense oligonucleotides (ASOs), I’m exploring the use of different conjugates for improving both delivery and target specificity. I’m particularly interested in learning from others in the field about:
  1. Which ASO conjugates (e.g., GalNAc, peptides, antibodies, polymers, etc.) have shown the highest level of target specificity in current research?
  2. Are there any conjugates that are proving to be more versatile and applicable across multiple targets or tissues?
  3. What factors are driving the choice of conjugates in your current research (e.g., target accessibility, therapeutic area, delivery challenges)?
I’d appreciate any insights or examples from your work or studies you’ve come across, especially those that demonstrate promising results in either highly specific or more universal delivery of ASOs.
Relevant answer
Answer
Deat Anna
This requires a long discussion. In summary, the two parameters of the route of administration and the target organ should be considered. For example, the use of galactose derivatives or lipid nanoparticles (not attached to other ligands) in IV injection will be useful when the target is the liver.
  • asked a question related to Antibodies
Question
1 answer
Does anyone have a good antibody for Fsp1 (S100A4) that can be used for IHC on frozen sections? Thanks so much!
Relevant answer
Answer
You can use a ready-to-use pathnsitu antibody; it will yield good results.
  • asked a question related to Antibodies
Question
7 answers
Hello all,
I am currently doing Dot Blots to confirm that my antibody is present after an Immunoprecipitant run and for some reason my signal is coming through in the FT and Wash but not in the Elution. My colleague and I are hypothesizing that the antibody we are testing for is stuck to the beads that we are using (AVIDIN for the 1st antibody and HRP for the 2nd) but we are not quite sure if this is the case or if something else is occurring. The kit we are using for Immunoprecipitating is a Thermo Pierce Classic IP kit. We don't know what else to do as we have been tweaking things like the pH for the Elution Buffer and we are kind of at a loss. The next thing we are going to try doing is modifying the wash agent by using 1x PBS but we aren't sure if that will solve anything. Does anyone have insight to this problem or perhaps a similar experience? Sorry this is my first time using ResearchGate so I hope my question comes across clearly.
Relevant answer
Answer
So I tried using 1xPBS with our method and unfortunately it yielded unsatisfactory results. I will try modifying the procedures with the additional suggestions provided and see if anything is yielded next week.
  • asked a question related to Antibodies
Question
2 answers
a small protein (~5KDa) is tagged with HA chromosomally. On doing western blotting and probing blot with anti HA antibody, the protein is showing band at ~13 kDa whereas expected size is ~6 kDa. Resolving gel used is 15%. How to resolve this problem?
Relevant answer
Answer
Any addition of negatively charged amino acids can lead to a shift to higher apparent MW in SDS-PAGE. The shift can be significant, especially in smaller proteins where the total negative charge is increased by 1 or 2 amino acids significantly. In the HA-tag you have two aspartates which could in addition to the size of the tag itself lead to a 13 kDa band.
Alternatively the protein could be dimeric in the gel although you would expect at least a weak band in the expected size.
I would say the first explanantion is more likely if you see only one band.
  • asked a question related to Antibodies
Question
1 answer
I have modified the paper with sodium periodate for covalent immobilization of my target protein. However, even in the absence of that protein, I get high-intensity colour by just adding the Enzyme-tagged detection antibody and its substrate. I wash the detection antibody with 1X PBST. My washing procedure is similar to that mentioned in most literature sources. I drop 10 uL of PBST on the test area (6mm diameter) and then remove excess water using a blotting paper on the bottom side of the paper. The paper I am currently working on is Type-1 Chromatography paper.
Relevant answer
Answer
What is the mechanism of your antibody colouring?
Do you wash away the sodium periodate? Maybe you're just immobilizing your antibody.
  • asked a question related to Antibodies
Question
2 answers
I am working on a project involving tissue-resident memory T cells in the thyroid, and CD69 is a key marker for these cells. However, I’ve struggled to find a CD69 antibody that works effectively for immunohistochemistry on paraffin-embedded (IHC-P) mouse thyroid tissue. I am particularly interested in antibodies that have been validated in similar studies. If anyone has experience with a specific CD69 antibody for this application, your insights would be extremely helpful.
Relevant answer
Answer
Please take a look at clone H1.2F3.
PE anti-mouse CD69 Antibody
  • asked a question related to Antibodies
Question
1 answer
Hello everybody,
I'm developing some truncated versions of ROR1 for in vitro studies and I would like to check by flow the absence of signal when targeting ROR1. I know that 2A2 binds to the N-ter, and most likely this means the Ig-like domain, but I don't have any clue whether exists an antibody (commercially available) against Frizzle or Kringle domains.
Does anyone know what epitope is each of these clones binding to? Is there any webpage where I can get this info?
Thank you very much in advance for all your help.
Relevant answer
Answer
When developing antibodies against truncated versions of ROR1 and checking signal absence by flow cytometry, it is crucial to understand the domains of ROR1 and their corresponding antibodies.
ROR1 structure contains several functional domains:
- Ig-like domain: This is the N-terminal part of ROR1 and is usually associated with the reception of extracellular signals.
- Frizzled (Fz) domain: This domain is associated with the Wnt signaling pathway and is involved in intercellular signaling.
- Kringles domain: This domain is commonly found in various proteins and may be associated with extracellular matrix binding and cell migration.
Antibody binding site: According to existing research data, monoclonal antibodies (mAbs) against ROR1 mainly recognize epitopes in the following two regions:
1. Ig-like domain:
- Most antibodies, including 2A2, mainly recognize the Ig-like domain of ROR1. This indicates that the binding site of 2A2 with ROR1 is likely to be located in the N-terminal region, which is very important in flow cytometry because it can effectively label and detect the expression of ROR1.
2. Frizzled and Kringles domains:
- Regarding antibodies against the Frizzled domain, although there is no clear indication in the literature that there are specific antibodies, studies have shown that the Frizzled region of ROR1 also has binding ability with other antibodies such as R12, which may target epitopes in the Frizzled region [2][3].
In flow cytometry, antibodies against different domains of ROR1 can help us better understand the role of ROR1 in cell signaling. By comparing the binding properties of other antibodies, the importance of different domains of ROR1 in signaling can be evaluated. For example, the 2A2 antibody has been shown to effectively block Wnt5a-induced ROR1 signals, which further supports the key role of ROR1 in cell migration and proliferation [6][7].
- Regarding antibodies against the Kringel domain, there is no direct mention of specific antibodies against this domain in the current literature. Nevertheless, studies have shown that different domains of ROR1 may be recognized by different antibodies, which provides a possible direction for the future development of antibodies against the Kringel domain [1][4]. For example, existing anti-ROR1 antibodies show different binding properties and effects, which may be related to the specific epitopes they bind to[4][5].
References:
[1] T. Tran. Armored tgfβriidn ror1-car t cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced tgfβ1, Journal for Immunotherapy of Cancer, vol. 12, no. 4, p. e008261, 2024. https://doi.org/10.1136/jitc-2023-008261
[2] M. Hudecek, M. Stanghellini, , et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ror1-specific chimeric antigen receptor t cells, Clinical Cancer Research, vol. 19, no. 12, p. 3153-3164, 2013. https://doi.org/10.1158/1078-0432.ccr-13-0330
[3] J. Qi, X. Li, et al. Potent and selective antitumor activity of a t cell-engaging bispecific antibody targeting a membrane-proximal epitope of ror1, Proceedings of the National Academy of Sciences, vol. 115, no. 24, 2018. https://doi.org/10.1073/pnas.1719905115
[4] R. Mani, Y. Mao, et al. Tumor antigen ror1 targeted drug delivery mediated selective leukemic but not normal b-cell cytotoxicity in chronic lymphocytic leukemia, Leukemia, vol. 29, no. 2, p. 346-355, 2014. https://doi.org/10.1038/leu.2014.199
[5] L. Aghebati‐Maleki, V. Younesi, et al. Antiproliferative and apoptotic effects of novel anti-ror1 single-chain antibodies in hematological malignancies, Slas Discovery, vol. 22, no. 4, p. 408-417, 2017. https://doi.org/10.1177/2472555216689659
[6] M. Hasan, L. Rassenti, G. Widhopf, J. Yu, & T. Kipps. Wnt5a causes ror1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells, Leukemia, vol. 33, no. 3, p. 653-661, 2018. https://doi.org/10.1038/s41375-018-0306-7
[7] P. Janovská, L. Poppová, et al. Autocrine signaling by wnt-5a deregulates chemotaxis of leukemic cells and predicts clinical outcome in chronic lymphocytic leukemia, Clinical Cancer Research, vol. 22, no. 2, p. 459-469, 2016. https://doi.org/10.1158/1078-0432.ccr-15-0154
  • asked a question related to Antibodies
Question
9 answers
Dear all,
I'm putting together an antibody panel to characterise airway epithelium.
I've tried two different p63 antibodies which show mostly unexpected cytoplasmic staining. I would expect 15-30% of epithelial cells to be positive with nuclear staining.
Has anyone else come across cytoplasmic staining in airway epithelium?
Details as follows:
FFPE section of lung tissue. Antigen retrieval done on Leica Bond Rx, blocked in BSA with Dk serum, Triton X-100 and Tween 20.
C1 (blue): DAPI
C2 (green): Ms x p63 (ab735, 1:100)
C3 (red): Rb x p63 (ab124762,1:200)
C4: merge
Thanks!
Relevant answer
Answer
It seems like you're using a different antibody to those that I've been using. My fixation times were 10 minutes, my cultures are only a few cell layers thick, so they don't require a long fixation time!
  • asked a question related to Antibodies
Question
5 answers
Hello everybody,
i'm currently developing an homemade sandwich ELISA. Currently to run such assay i require three days (1 plate derivatization, 2 blocking and sample incubation , 3 detecion)
I was wondering, can derivatized plate with capture antibody be stored at 4°C? If so, which condition are better, dry or i need some storage buffer to prevent contamination and antibody denaturation?
Currently for the assay i use MaxiSorp plates, and the derivatization conditions are over-night incubation at 4°C with 100uL/well of capture antibody (0,2 ug/mL).
Thank you in advance for your kind answer
Relevant answer
Answer
Go a head
  • asked a question related to Antibodies
Question
2 answers
As we know that antibodies are the products of an antigen invasion during humoral immune response. So where does the anti-B antibody come from for a type A blood person when no transfusion occurs?
Relevant answer
Answer
Malcolm Nobre Thank you so much for the answer which makes big sense to me.
  • asked a question related to Antibodies
Question
3 answers
Hi all,
Recently I have been having trouble exposing my tubulin. There seems to be an uneven exposure of the lanes, yet my ponceau looks even across. From what I have read online, this could possibly be due to uneven antibody exposure, however I use the same method for all my antibodies and am only having trouble with tubulin.
My methods:
1. After transfer, add blocking buffer (depending on antibody: %5 milk or 5% BSA) for 1 hr
2. Prepare primary antibody solution at 1:1000 in 1x TBS-T. Incubate membrane overnight on roller.
3. Wash three times for 5min each (total 15min) with 1x TBS-T.
4. Prepare secondary antibody at 1:2000 in 1x TBS-T. Incubate 4C for two hours on roller.
5. Wash three times for 5min each (total 15min) with 1x TBS-T
6. Prepare ECL solution at 1:1. Incubate membrane for 1min each and then expose using film/developer
My troubleshooting so far:
1. Buy new non-fat milk (ours was expired)
2. Decrease/increase antibody concentrations
3. Apply ECL directly to membrane and expose immediately (to avoid signal being lost quickly)
4. Prepare fresh antibody every time
Any tips would be helpful. Attached is a picture for reference.
Relevant answer
Answer
Malcolm Nobre Koen van Wijk thank you both for your responses! I did try reducing the secondary antibody as well as placing the membranes on a shaker instead of in tubes on a roller. I have had luck so far with these methods but I will also try my secondary at room temperature.
Koen, thank you for the tip about the ECL, I have seen others do this but have never tried so I will give it a shot. This is the first lab I have been in that uses a developer, but I enjoy the old school!
  • asked a question related to Antibodies
Question
3 answers
I am basically working with self-assembled VLPs. I am not sure if His tag in my protein is not exposed (I can detect in reducing western blot by his tag antibody) or other technical issues, and most of my protein appear in flow through. I used pH of 8 for buffer. Should I wash with several column volume of buffer before starting? Thank you for any other suggestions!
Relevant answer
Answer
Yes, washing with several column volumes of buffer before loading the sample is a good idea to equilibrate the resin. Other suggestions:
pH 8 is fine, but ensure it doesn’t exceed 8.0. If binding is weak, try lowering the pH slightly (7.4-7.6).
Keep the imidazole concentration low (~10-20 mM) during the binding phase to reduce non-specific interactions but allow proper binding.
Allow the sample more time to bind to the resin (30-60 min at 4°C).
  • asked a question related to Antibodies
Question
1 answer
I have stained PBMCs with fluorescently labelled antibodies and fixed them for flow cytometry. I know they should be stored at 4oC until analysing, but will they be okay left at room temperature for 6 hours or is this too long? Thanks!
Relevant answer
Answer
Hello Lucy B.
Not sure. Once you have stained the cells, there are certain points you need to consider for how long the fluorochrome will remain.
1. Cells die with time and antibodies are internalized gradually at warm temperatures which can produce a loss of fluorescence intensity.
2. The fluorochromes become bleached in light (you didn’t mention whether the stained PBMCs were in the dark or in light at room temperature).
3. There would be microbes growing eventually.
So, keep cells (before and after staining) at 4°C and in the dark. Use a metabolic poison like azide in all buffers since the presence of sodium azide will prevent the modulation and internalization of surface antigens, if you are not concerned with recovering cell function, and, finally, fix the cells with formaldehyde after staining if you are not going to analyze them within several hours on flow cytometer.
For best results, analyze the cells on flow cytometer as soon as possible. Whether the fixed cells stained with fluorescently labelled antibodies would be okay at room temperature for 6 hours, would be difficult to answer, unless you go ahead and carry out the analysis.
Best.
  • asked a question related to Antibodies
Question
7 answers
Hi. I'm analyzing the kinetics of antibody antigen interaction. I have some that show very low Koff. The problem is in the dissociation phase the curve is not only stationary but increase....the reviewer need explanation. Is a question of normalization with the base-line? I mean there are not many things I can have done a wrong
in the experiment. Please I need a acceptable justification.
Relevant answer
Answer
Hi Alexander! How did you solve the problem? I’m facing similar problem, where the dissociation curve is a straight line, instead of dropping back to baseline. I tried increasing the dissociation time as well but didn’t help much.
I’m using octet rh16 to study two proteins’ interactions.
  • asked a question related to Antibodies
Question
2 answers
I am using silver staining for an experiment. Lanes 2 and 3 are rabbit serum containing my Antibody of interest under reduced (DTT) and non-reduced conditions. Lane 2 always turns yellow no matter how long I incubate with developer solution. What could be the reason for this turning yellow? Could someone help me with this.
Relevant answer
Answer
excess DTT can cause this if the DTT concentration is over 50mM
Use 30–50 mM DTT for reducing the sample
  • asked a question related to Antibodies
Question
2 answers
Dear colleagues, I would like to specifically purify an anti-HLA-A2 antibody from plasma samples of a kidney transplant patient. I believe that antigen-specific affinity purification might be the best option, but I'm not entirely sure. Does anyone have experience with this process? Thank you very much!
Relevant answer
Answer
Yes, antigen-specific affinity purification is a widely used technique to isolate antibodies that bind specifically to a particular antigen from a mixture. Here’s a detailed protocol for this process:
### **Protocol for Antigen-Specific Affinity Purification of Antibodies**
#### **Materials:**
1. **Antigen (for immobilization)**
2. **Affinity matrix** (e.g., Protein A/G beads, agarose or sepharose beads)
3. **Antibody-containing sample** (e.g., serum, cell culture supernatant)
4. **Binding buffer** (e.g., PBS or TBS)
5. **Washing buffer** (e.g., PBS or TBS with a small amount of detergent like Tween-20)
6. **Elution buffer** (e.g., low pH elution buffer, such as 0.1M glycine-HCl, pH 2.5-3.0)
7. **Neutralization buffer** (e.g., 1M Tris-HCl, pH 8.0)
8. **Centrifuge**
9. **Microcentrifuge tubes**
10. **Column (if using a column-based system)**
#### **Procedure:**
**1. Prepare Antigen-Coated Affinity Matrix:**
- **Couple Antigen to Matrix:**
1. **Choose the matrix**: Common matrices include agarose or sepharose beads. For most applications, Protein A/G beads are also used.
2. **Prepare the matrix**: Swell the beads in the coupling buffer (usually a carbonate-bicarbonate buffer, pH 9.0, for antigen coupling).
3. **Couple the antigen**: Incubate the antigen with the matrix according to the manufacturer's instructions. Typically, this involves incubating the antigen and beads overnight at 4°C with gentle agitation.
**2. Block Unreacted Sites:**
- After coupling, block any remaining reactive sites on the matrix to prevent non-specific binding. Use a blocking solution, such as 1-5% BSA in PBS, and incubate for 1-2 hours at room temperature or overnight at 4°C.
**3. Wash the Matrix:**
- Wash the matrix with several volumes of PBS or TBS to remove any unbound antigen or blocking agent.
**4. Bind Antibodies:**
- **Add Antibody Sample**: Incubate the antibody-containing sample with the antigen-coated matrix. This is typically done by gently mixing the sample with the matrix and incubating at 4°C for 1-2 hours or overnight with gentle agitation.
- **Volume and Concentration**: Adjust the volume and concentration of the antibody sample according to the amount of antigen on the matrix and the expected antibody concentration.
**5. Wash:**
- After binding, wash the matrix with multiple volumes of washing buffer (e.g., PBS or TBS with 0.05-0.1% Tween-20) to remove non-specifically bound proteins. Perform several washes until the flow-through is free of unbound antibodies.
**6. Elute Specific Antibodies:**
- **Elution**: Elute the specifically bound antibodies using an elution buffer. Typically, this is done with a low pH buffer, such as 0.1M glycine-HCl, pH 2.5-3.0. Collect the eluted fractions in microcentrifuge tubes.
- **Neutralize**: Immediately neutralize the eluted antibodies with a neutralization buffer (e.g., 1M Tris-HCl, pH 8.0) to prevent denaturation or damage to the antibodies.
**7. Concentrate and Store:**
- **Concentration**: If needed, concentrate the antibody solution using a centrifugal concentrator or other suitable methods.
- **Storage**: Store the purified antibodies at -20°C or -80°C for long-term storage. For short-term use, they can be kept at 4°C.
**8. Validate Purity:**
- Verify the purity and specificity of the purified antibodies using techniques such as SDS-PAGE, Western blotting, or ELISA.
### **Notes:**
- **Antigen Coupling Efficiency**: Ensure that the antigen is coupled efficiently to the matrix by optimizing the coupling conditions.
- **Matrix Choice**: The choice of matrix can affect the binding and elution conditions. Always follow the manufacturer's recommendations.
- **Buffer Conditions**: Buffer composition can significantly impact the efficiency of binding and elution. Adjust buffers based on the specific properties of your antibodies and antigens.
By following this protocol, you should be able to perform antigen-specific affinity purification effectively, obtaining purified antibodies with high specificity to your target antigen.
  • asked a question related to Antibodies
Question
4 answers
Dear all,
I'm supposed to radiolabel pembrolizumab and nivolumab. Antibodies are pretty expensive and I would like to pratice (reaction /purification/ QC) on an antibodie modele instead of using the more expensive ones ?
What do you suggest ?
Thanks a lot.
Relevant answer
Answer
Manuele Martinelli thank you so much.
My best reagrds
Laetitia
  • asked a question related to Antibodies
Question
4 answers
I'm stimulating CD4+ T cells for 3 and 5 days with plate-bound CD3 antibody, soluble CD28 antibody, and LPS. I want to measure intracellular cytokine levels on days 3 and 5 (IL4, IFNγ, IL10, and IL17A) via intracellular staining and flow cytometry. When should I add brefeldin A to my cells in order to detect intracellular cytokines? 4 hours before analysis? 12 hours? Any help would be appreciated.
P.S. I've already analyzed T cells stimulated for 3 days for IL17A. I added brefeldin A 4 hours before analyzing the cells, but I didn't see any IL17A+ T cells. This is making me worry that perhaps I didn't add BFA add the optimal timepoint.
Relevant answer
Answer
Thomas Ching-Jen Tan This is exactly the kind of information I was looking for. Thank you! I'll try stimulating for 48 hours and then restimulating my CD4 T cells after their expansion.
  • asked a question related to Antibodies
Question
2 answers
We have tested the binding of an antibody with its target (antigen) using gel shift assay, ELISA and SPR. Theorectially the antibody should bind to its antigen in all three assays. However, it turned out the results were only positive in ELISA and SPR but not in gel shift assay. Did you happen to see the same phenomenon?
Relevant answer
Answer
We did not run EMSA. The antigen is a protein.
We ran the binding assay using size exclusion chromatography (SEC), also called gel filtration. If the antibody binds to the antigen, in SEC we should see the peak shifts, both antibody and antigen peaks decrease and antibody-antigen peak increases. However, we did not see these shifts happened.
We did see the binding in ELISA and SPR.
  • asked a question related to Antibodies
Question
8 answers
I am planning to order an antibody, but for the commercially available antibody, it is written that the application is ELISA. So, if I want to order the same antibody can we use that?
Relevant answer
If the antibody comes with the ELISA kit, better to avoid using it. Since it has probably been diluted to cater for ELISA analysis. Also, usually Western blot need a higher concentration of antibodies. But it’s possible if it’s the other way around.
  • asked a question related to Antibodies
Question
1 answer
Hi All,
I am ordering an overalapping peptide library to study the binding epitope of my antibody. I wonder if there is a formula to calculate the probability of number of epitope hits (e.g. single, double) with different epitope length, peptide length and offset (or peptide overlap). Knowing the probability of double hit would be helpful to determine how many peptides to order (as they are quite expensive!). Thank you for your help.
Relevant answer
Answer
I did a *lot* of this kind of work as a post-doc, when multiple peptide synthesis was first becoming feasible. There is no easy answer to your question, as asked.
What do you know about your Ab, and what do you need to know about the epitope?
If you have a monoclonal there is about a 12% chance it will bind to a short peptide. Most MAb are specific for conformational epitopes, and it's infamously difficult to draw conclusions about their epitopes using short peptides as antigens. OTOH, if you're looking at antiserum, you're likely to find multiple epitopes, with one or two immunodominant.
What level of resolution do you need? If you plan to follow up with more synthesis and mapping you can get away with an initial experiment using fewer peptides (e.g., 12mers overlapping by 4).
Anyway, yes, making the peptides is a lot of work and expensive. So once you have them, test as many Ab as you can find.
  • asked a question related to Antibodies
Question
8 answers
I have an bispecific antibody which was stable expressed by 293F cell, but degraded seversely expressed by ExpiCHO-S cells, I wonder if there is any chance to optimize and to obtain stable antibody expressed by ExpiCHO-S, such as to change the linker, or harvest cell a few days earlier
Relevant answer
Answer
Depending on the antibody format, degradation does happen. I have faced the same issue, I have expressed some scFv-Fc molecules in 293 and when I switched to CHO I started seeing cleavage. Interestingly, the cleavage was right between the scFv and Fc, generating same size fragments. I have realized that the hinge my affect cleavage. What hinge sequence have you used to connect your scFv and Fc?
Have you tried using protease inhibitors after expression?
  • asked a question related to Antibodies
Question
9 answers
So I used CRISPR-Cas9 system to knock out the protein of interest. I sequenced the modified region, and then I analyzed and there are 2 KO clones according to my results (deletion-causing stop codons). Next step was western blot to detect the protein level, but when I did the chemiluminescence detection and applied long exposure time I got faint bands- 2 faint bands... I think my antibody is not perfect, because it is a polyclonal antibody.. might be same other protein in that band, beacuse my antibody is not so specific and it could recognise other proteins and bind to them?? What do you think? I checked my clones with RT-qPCR too, and I got amplification in the KO clones too, same as the wild type... what do you think about this? I am confused and try to find out a good answer to this phenomenom.. my ideai is the mRNA is transcribed and the (mutated) protein is traslated , but after that it is degradated, because the protein is truncated or the folding is not good.. but why I see faint bands? might be the antibody? What should I do?
I am looking forward your theories and answers.. Thank you so much :)
Loretta
Relevant answer
Answer
Basics of CRISPR-Cas9: How it works and its components.
Designing gRNAs: Tips for designing effective guide RNAs.
Applications: Different uses in research or therapy.
Experimental Setup: How to set up a CRISPR experiment.
Troubleshooting: Common issues and how to solve them.
Ethical Considerations: Discussions on the ethical use of CRISPR technology.
l Check out this protocol list; it might provide additional insights for resolving the issue.
  • asked a question related to Antibodies
Question
3 answers
I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity chromatography to select the antibodies.
However, I haven't found any articles or protocols that describe using this column in this specific context.
Has anyone used the HisTRAP column for this particular purpose? If so, which buffer did you use to elute only the antibodies without eluting the bound protein?
Relevant answer
Answer
Hi Jackelinne,
I don't think it will work well. First, antibodies themselves can bind on NiNTA (generally IMAC was developed for purification of natural non tagged proteins, even before his-tag). Secondly elution, highly acidic conditions will be required to remove antibodies from the antigen. At low pH the recombinant protein itself will elute from the column (firstly histidines are protonated and secondly nickel stripping occurs). Part of the problem with nickel stripping can be circumvented if you use Sepharose Excel. Nickel binds firmly there, but the binding of recombinant protein to the nickel histag will weaken anyway.
It is easier to immobilize the recombinant protein on Sepharose by any suitable method (cyanogen bromide activation, epoxyactivation - there are even ready-made activated resins) and purify the antibodies in this way.
  • asked a question related to Antibodies
Question
4 answers
Can anyone share any reference that gives mouse specific CD3 antibody sequence. I have been searching for this since few days and no luck. Thank you very much
Relevant answer
Answer
This may be a difficult information to obtain, as usually the antibodies are not characterized in their protein sequence.
But if the antibody is a monoclonal, you may characterize it yourself.
Why do you need this information?
  • asked a question related to Antibodies
Question
3 answers
We have a lateral flow test on Sartorius 140 NC. The conjugate is gold-monoclonal antibody. Using different control lines (GAM, protein A, protein G) we get a very strong leading edge with reduced signal across the 1 mm of the line. We have tried different concentrations of everything and different striping buffers. Is there a way to make the line more uniform?
Relevant answer
Answer
Thanks for the replies Motasim Mohammedahmed and Ishrat Jahan Saifi .
We've tried pretty much all of your suggestions. We're using the best quality papers and reagents. I read somewhere that this leading edge effect may be intrinsic to the affinity of the antigen/antibody combination we are using and cannot change. We don't have the same problems with other tests. Thanks for your suggestions!
  • asked a question related to Antibodies
Question
4 answers
I would like to enhance the fluorescent signal of mRuby3 in brain slices. I have only found antibodies good for WB. Did anyone try them on tissue? Is there a good antibody for IHC?
Relevant answer
Answer
Hi Anna. Have you solved this problem?
I wonder if sdAb would work. Have you ever tested sdAb?
Thanks!
  • asked a question related to Antibodies
Question
3 answers
Hello,
I'm looking at immunoprecipitating our flag tagged protein in a chondrocyte cell line derived from mouse. So I need recommendations for a good IP antibody for flag produced in rabbit that could also be used for the subsequent western blot. Any recommendations would be much appreciated. Thank you.
Relevant answer
Answer
I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
A highly recommended rabbit-produced antibody for immunoprecipitation (IP) and subsequent western blotting of FLAG-tagged proteins in mouse-derived chondrocyte cell lines is the Anti-FLAG® M2 antibody from Sigma-Aldrich. This antibody demonstrates high affinity and specificity for FLAG-tagged proteins, making it suitable for capturing and detecting these proteins in various applications, including IP and western blotting [1][2][3][4]. The Anti-FLAG® M2 monoclonal antibody is widely used due to its ability to bind both N-terminal and C-terminal FLAG tags without requiring calcium-dependent conditions, ensuring reliable and consistent results [5][6].
Reference
[1] Brizzard, B., Chubet, R., & Vizard, D. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution.. BioTechniques, 16 4, 730-5 .
[2] Knappik, A., & Plückthun, A. (1994). An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.. BioTechniques, 17 4, 754-61 .
[3] Ferrando, R., Newton, K., Chu, F., Webster, J., & French, D. (2015). Immunohistochemical Detection of FLAG-Tagged Endogenous Proteins in Knock-In Mice. Journal of Histochemistry & Cytochemistry, 63, 244 - 255.
[4] Verhagen, A. (2006). Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins.. CSH protocols, 2006 5.
[5] Itakura, E., Chen, C., & Bono, M. d. (2017). Purification of FLAG-tagged Secreted Proteins from Mammalian Cells.. Bio-protocol, 7 15.
[6] Zhang, L., Uder, S., Juehne, T., Brizzard, B., & Song, K. (2002). Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates.. BioTechniques, 32 2, 442-7 .
  • asked a question related to Antibodies
Question
4 answers
Hi,
I'm working on heart flow cytometry, and I use anti-mouse CCR2-BV785 (biolegend, cat150621) for staining, 20 minutes, 4 degree. But I couldn't see any CCR2+ cells. So I'm not sure if it's actually right or it's because of antibody not working. I just checked some literature and it's supposed to have more CCR2+ cells in heart failure.
Do you have any ideas?
Thank you so much.
Best,
Relevant answer
Answer
Tobias Kammann Thank you. Did you wash cells after CCR-specific antibodies intubation? Or just add the rest of antibodies?
  • asked a question related to Antibodies
Question
10 answers
i am trying to stain different proteins of interest in human paraffinized section.
my signal should be the vessel only, but regardless of the antibody I get circle shaped spots , I tried antigen retrieval with trypsin, and I tried different dilution of antibodies (1:100-1:1000) and blocking in 5% and 10% donkey serum
why I have those spots? how can I reduce them? I have the same issue at different wave lengths (regardless of secondary antibodies tag)
this is my protocol:
•Thickness of sections : 10µ
•Deparaffinization by heat 55degrees for 20 min then (xylene - xylene: ethanol - ethanol) 10min each*twice
•Hydration (ethanol 95% - 70% - 50% - tab water) 5min each*twice
•Antigen retrieval : citrate buffer 10min microwave then leave in buffer to cool
•Peroxide treatment: 3%H2O2 10min at room temp
•Blocking: 5% Donkey serum + 0.3% Triton-PBS 1 hr RT
•Antibodies:
•Primary in 1%BSA: VWF (1:200)
•Secondary 1:500 of Rabbit 594
Relevant answer
Answer
Hello all,
I found this interesting paper in which authors described their protocol to get rid of the background in their staining.
Hope it can be helpful!
  • asked a question related to Antibodies
Question
6 answers
I am performing IgG purification and I have to show my results on SDS-PAGE. I use 10% tris glycine gel and prepare the samples under non reducing conditions. I am new to antibodies and therefore need some help. First of all, my main band in cell lysate is about 50 kDa for some reasons. Purified antibody showed the same band and also one at around 250kDa.
Initially, I did reducing conditions and got one extra band at around 25 kDa in elute, so I changed to non-reducing. I attached SDS-PAGE gel (non-reducing), L- lysate, E- elute, M- marker (10-250 kDa)
Relevant answer
Answer
The concentration of albumin in culture media containing serum is quite high. There are other proteins, too, but albumin is by far the most abundant. It can be difficult to get rid of all of it when purifying IgG from culture medium. There are resins available to help remove it, or you may be able to adapt the cells to grow in serum-free medium, which lacks albumin.
You mentioned a lysate, suggesting that the IgG was produced within the cells rather than excreted into the medium. If this is the method of production, thoroughly washing the cells to remove external albumin before preparing the lysate would probably help. Also, make sure there is no albumin in the lysis reagent, of course.
  • asked a question related to Antibodies
Question
1 answer
Hello,
I recently stained my cells with an Arf6 antibody. The healthy control looks pretty normal, while the disease group cells have abnormally large black structures in the cell body. Has anyone experienced anything similar? Are those holes, vesicles, or anything else??? For further info, those cells are destined to die, so they might as well be disintegrating at the fixation time. Still, I am curious about what I saw.
Relevant answer
Answer
Elif Bayraktar , 'digestion', 'lipidic' or 'enzymatic' 'vacuoles' ["...ARF genes...play a role in vesicular trafficking and as activators of ....phospholipase D."] come to my mind immediately. Unfortunately I haven't seen such cells in culture stained by Arf6 (just for the record and for correct 'RG-archive search phrase" reasons: Arf6= ADP-ribosylation factor 6) Antibody.....
But: I don't think it were 'disintegrating, previously nuclear' bodies....
To get proof of the nature of those "holes" or "black structures" (as you named them), in an attempt of correlative light microscopy, proper chemical fixation and processing at least into paraffin wax (if you have options for and would think of cryofixing / cryoprocessing, staining, etc....you might try...but I'd guess, the results wouldn't reveal the "real nature" of the morphological conspicuities) seems mandatory.... To achieve optimal results IMHO one would perform classical specimen preparation for TEM-observation on the ultrastructural scale...
  • asked a question related to Antibodies
Question
6 answers
Hi there, I'd like to use immunocytochemistry to determine a surface protein expression on mouse cell. The primary antibody I have is mouse anti mouse, and the secondary antibody I have is goat anti mouse which is conjugated with fluorochrome. Is there any problem with above antibodies selection? I would really appreciate it if someone could help me solve this problem.
Relevant answer
Answer
Manuele Martinelli Hi Manuele, actually I'm doing immuocytochemistry on the mouse cells. The technique of antibody labelling that you mentioned sounds so cool. I will take a look. Thank you so much for the information.
  • asked a question related to Antibodies
Question
2 answers
A vaccine is a shot of a disease into someone's body, to summon antibodies to fight the illness. If the exact and specific cause of aging could be identified then t cells may mass produce themselves for anti aging. First, aging, as a disease, must have an identified parsimonious cause.
Relevant answer
Answer
I think aging is not a disease while vaccine require microorganism or their product the cause of infection
  • asked a question related to Antibodies
Question
3 answers
Dear all,
I'm going to radiolabel antibodies. So first of all, I'll remove excipients using the centrifugation method with amicon.
Once I'm done, how do I calculate/measure the concentration of the antibody ? We don't have a nanodrop in the lab.
Thank you for your help
Laetitia
Relevant answer
Answer
if do you have any other UV-VIS spectrophotometer you can use it instead nanodrop to determine the A280nm and afterwards extrapolate the mab concentration considering that the molar exctinction coefficient of a mab is around 210,000M-1 cm-1.
Theoretically at 280nm, you have to use quartz cell to obtain reailable determination since standard plastic in not trasparent to UV but there are actually many commercial plastic cuvettes that has low absorbance at 280nm and therefore could be used. Just look to the absorbance of your empty cuvette before perform the experiment. If it is relativelly low (max0.1-0,2 you can proceed, if it is high eg. 0.5 or more) is it better to found a better cuvette
best
Manuele
  • asked a question related to Antibodies
Question
3 answers
I have a set of finished samples for immunofluorescence antibody analysis, but by applying the mounting medium a large number of microbubbles, appeares, would it be possible to replace it at this point?
Relevant answer
Answer
thank you so much!!!
  • asked a question related to Antibodies
Question
3 answers
I am trying to couple several antibodies (including antiCD14 and antiCD3) to magnetic nanobeads, after coupling they immediately form aggregates. Does anyone know why?
Relevant answer
Answer
Do you sonicate the nanobeads before using them? Many nanoparticles tend to aggregate and this usually helps.
  • asked a question related to Antibodies
Question
3 answers
I have this problem with my western blot for the ABCG2 antibody on nitrocellulose membrane. Help please
Relevant answer
Answer
Echoing others, what am I looking at? I see some sporadic dots on a membrane and one that appears to be completely black. Is this correct? Echoing the first two, the black membrane hasn't been adequately blocked, if at all. You'll want to use a solution of 5% milk in PBS with 0.1% Tween-20. Block AT LEAST 40 minutes. Longer for whole cell lysates and you'll want to just spike your primary into the blocking too. My personal experience is that this does help cut down on nonspecific binding.
As for the membrane that looks like it doesn't have any dots/bands (I think there is a membrane there), this could be caused by multiple things:
1.) Your primary antibody may be bad/old. Not all clones work for WBs. Check the manufacturer's website and make sure the one you're using is validated for WB use. Although the standard dilution for primary is 1:1000, you may need to do a titration to find if you need a higher or lower dilution to get a clean looking WB image.
2.) You did not incubate the membrane in primary long enough. Overnight at 4 C is what I've always done, although sometimes you can get away with RT for 2 hours.
3.) You used the wrong secondary antibody. If for example, if the host for your primary is mouse, make sure the 2nd antibody is anti-mouse.
4.) You may have reacted all the luminol (if you imaged for a while) and there is no more chemiluminescence for the imager to collect.
I hope this helps you! Good luck!
  • asked a question related to Antibodies
Question
1 answer
One realisation is that my protocol with 10%NGS in PBST is not working as well as the IC solution. I need to prepare that solution independently but I do not have the recipe. My protocol is fix-permeabilize-block-primary Ab in blocking overnight incubation- secondary antibody incubation- HOECHST and imaging. Washes are done in 1x PBS. In IC protocol, fix-2.5%NGS inactivated blocking in IC soln -primary ab in IC solution o/n incubation-secondary ab in IC soln- HOECHST in PBS- imaging. In IC protocol, washes are done in IC solution.
Both protocols were performed simultaneously on the same type of cells under the same conditions including the dilutions.
Relevant answer
I found a way to achieve better staining. I used saponin 0.1% PBS instead of triton.
Adding 3% normal goat serum and 2% bovine serum albumin in 1xPBS 0.1% saponin also gives beautifully stained cells.
  • asked a question related to Antibodies
Question
4 answers
I have tried staining the mouse brain free floating sections (4% PFA fixed) using CD31 antibody from different vendors and nothing has worked. I see beautiful staining in number of references but I could never get it. Any one has experience with this antibody?
Relevant answer
Answer
Dear Gulnaz Begum
I was trying to download this protocol (Final IHC Protocol for CD31_PK Antigen retrieval_Updated 111
7.docx) but the file was removed. by any chance do you still have it ?
thank you in advance
  • asked a question related to Antibodies
Question
7 answers
I need to do immunohistochemistry in mouse brain, particularly in neurotensin-cre mouse line.
Relevant answer
Answer
Hello Xiuzhen,
we are offering antibodies against all the targets you mention, so it would be a lot of work to align the epitopes between the immunogen and the gp sequence, so pleas econtact me via my e-mail address.
Best regards,
Carsten
  • asked a question related to Antibodies
Question
3 answers
I'm planning on activating 5 million PBMCs with CD3/CD28 at the same time applying a treatment, then incubate for 72 hours. Then I seperate cell subtypes using antibody sequencial seperation method. I initially thought of plating them in 150mm non-treated plates but now I'm second-guessing because many literature indicates plating PBMCs at 1 million cells per mL in 24 well plates or 6-well plates. will 150mm plates be too big and can affect the viability of the cells?
Relevant answer
Answer
Usually we plate 1.5 million of cells per well in 6 well plates (1 million cells/ml), if you want to plate 5 million of cells, I will use T25 culture flasks, which might be more convenient
  • asked a question related to Antibodies
Question
1 answer
Can anyone recommend polyclonal or monoclonal antibodies for
Tet methylcytosine dioxygenase 2 (TET2)
Tet methylcytosine dioxygenase 3 (TET3)
DNA methyltransferase 3 beta (DNMT3B)
Ideally, they should work on bovine tissues using immunohistochemistry methods on paraffin sections and western blots. We are interested in testing the corpus luteum of a cow. However, it is a tissue that contains some fat, which can hinder antibody binding. We have already tried several antibodies, and none of them worked. However, gene expression studies of TET2, TET3, and DNMT3B revealed their expression in this tissue.
Relevant answer
Answer
Not a personal recommendation, but GeneTex and Biorbyt have anti-TET3 pAbs that they say are bovine reactive:
and Millipore-Sigma has bovine reactive anti-DNMT3B:
  • asked a question related to Antibodies
Question
3 answers
I need an appropriate positive control for WB antibodies I am using to confirm the presence of exosomes in samples. The thing is I am having reproducibility issues, and I would like distinguish whether it is my sample that is the problem because it has no exosomes or if it is the antibodies that aren't working properly. Running a cell lysate of a cell known to express these markers would help me determine this.
Thanks to anyone who took the time to read this and reply.
Relevant answer
Answer
several lymphocytes populations express tetraspanins
  • asked a question related to Antibodies
Question
2 answers
Recently, I have faced an issue with my antibody not picking up my band of interest correctly. Previously, this antibody has worked with no issues at all (expected blot) and has produced beautiful clean bands, but some months ago, the same antibody started to blot really blotchy bands. Surprisingly, my beta-actin blots perfectly. I have reordered the same antibody but it still behaves the same way even though the lot numbers of the antibody are different.
I have tried running my gel slower and at a lower voltage, doing a 2h primary antibody incubation/1h secondary incubation, leaving the primary on overnight, using both ECL and West Femto and making new aliquots of my primary. None of these have worked in my favour.
Is there anything else that I can try?
Relevant answer
Answer
Hello Ruchi,
I disagree with Dr. Iskander Madhi, i don't think this is an issue of protein quality. I think its related to how you are incubating your membranes with antibodies. They must be kept at 4 degrees with continuous, gentle shaking (I am speculating here that you aren't keeping your membranes for antibody incubation on a shaker). Also, I would like to know the size of the protein of interest. If you are using a shaker, then it could be something else, cannot say for sure, until we get to know the protocol you are following.
  • asked a question related to Antibodies
Question
1 answer
Hey everyone! I'm having difficulty marking the C4 complement factor in Western Blotting. Has anyone had contact with this antibody or made it in-house? I have already used Anti-C4β (D-12) sc-74524 from Santa Cruz Biotechnology.
Thank you!
Relevant answer
Answer
Hey there! I understand your challenge with marking the C4 complement factor in Western Blotting. Here are a few suggestions that might help:
  1. Antibody Validation: Ensure that the Anti-C4β (D-12) sc-74524 antibody from Santa Cruz Biotechnology is validated for Western Blotting. Sometimes, antibodies might work well in one application but not in others.
  2. Antibody Concentration: Adjust the concentration of the antibody. Sometimes using too much or too little can affect the binding and signal.
  3. Blocking Conditions: Optimize your blocking conditions. Try different blocking agents like BSA or milk, and different blocking times and temperatures.
  4. Secondary Antibody: Check your secondary antibody. Make sure it is compatible with your primary antibody and that it is functioning properly.
  5. Sample Preparation: Ensure that your sample preparation is done correctly. Proper lysing of cells and correct loading amounts are crucial.
  6. Incubation Times and Conditions: Optimize incubation times and conditions for both primary and secondary antibodies. Sometimes longer or shorter incubation times or different temperatures can improve results.
  7. In-House Antibody: If you've considered making the antibody in-house, ensure you have access to the proper equipment and expertise. Producing high-quality antibodies requires precise techniques and conditions.
  8. Alternative Antibodies: Consider trying alternative antibodies from different suppliers. Sometimes, an antibody from a different source might work better for your specific application.
  9. Technical Support: Contact the technical support team of Santa Cruz Biotechnology. They might provide additional tips or troubleshooting specific to the antibody you're using.
  • asked a question related to Antibodies
Question
3 answers
We aim to detect p62 protein expression levels. Previously, we worked with the p62 antibody (Cat. No: A19700, dilution 1:1,000, ABclonal) and obtained excellent results. However, we switched to a new antibody (p62 Antibody, sc-48402, dilution 1:1,000, Santa Cruz Biotechnology), and our bands showed excessive background noise. We tested different dilutions (1:1,000, 1:2,000, and 1:4,000), but the background noise did not decrease.
Any advice would be greatly appreciated.
Relevant answer
Answer
Go back to the original antibody. If it is no longer supplied then ask abclonal where the original source of the antibody came from and contact that company to buy the one that works well
  • asked a question related to Antibodies
Question
1 answer
Hello, could you please give me precise details of the antibodies that can be used to perform quantitative immunofluorescence of AT1Rs in the mouse brain?
Relevant answer
Answer
Antibodies produced i rabbits please follow of binding of Herolog ous anti brain antibodies to mouse B cells
  • asked a question related to Antibodies
Question
3 answers
Hi,
I want to analyze the protein expression levels of HPV16 E6/E7 in the cervical cancer cell lines SiHa and CaSki after treatment with silencing RNA for this oncogene. Can anyone recommend a suitable antibody for western blot analysis to detect these proteins?
Relevant answer
Answer
Choose antibodies that are specific for the HPV16 E6 and E7 proteins to avoid cross-reactivity with other proteins. Some antibodies are identified by a clone number which can indicate their specificity and performance in certain applications. The optimal concentration of the antibody for Western blot may vary, so it's important to follow the manufacturer's recommendations or perform titrations.
  • asked a question related to Antibodies
Question
3 answers
Hi everyone,
I wanna get a suitable protocol for depletion of IgG and other immunoglobulins from serum, without kits or chromatography but with chemical methods like ammonium sulfate. Can anyone help me? Does anyone have any experiences about it?
Relevant answer
Answer
Totally avoid reducing agents like 2-mercaptoethanol or dithiothreitol. Following SDS-PAGE under NON-reducing conditions, (heat for only a few min), IgG will land at 150 kDa far away from your target molecule.
  • asked a question related to Antibodies
Question
1 answer
Can anyone recommend high-quality GSDMD antibodies for immunofluorescent or immunohistochemical staining in mice?
Relevant answer
  • asked a question related to Antibodies
Question
5 answers
Dear all,
I have a question regarding the interpretation of Western blot results. I’m unsure why the line does not intersect the x-axis at zero on the graph. I’d like to seek your assistance in resolving this issue. Additionally, I’ve noticed that the dillution of secondary antibodies seems to affect the line’s position. Perhaps different antibody dilutions will result in the line intersecting the graph at zero.
The legend for the descriptions is as follows: 1 AB - primary antibodies, 2 AB - secondary antibodies, 1:2000, etc., antibody dilution
Relevant answer
Answer
Michael Ronzetti and Steven Dodd, many thanks for your comments.
  • asked a question related to Antibodies
Question
2 answers
Hi, I sometimes get these circles forming in my cell IF. They do not always show up, but sometimes I can't seem to avoid them even when I follow the same protocol. Any ideas what may be causing this issue?
The red signal is supposed to be cox4. I have included a reference photo to show the expected staining pattern.
Relevant answer
Answer
Perhaps there is an intermittent source of oxidative stress. See figure 2.
  • asked a question related to Antibodies
Question
5 answers
We produced scFv of an antibody from bacteria. Now we want to establish dissociation Kd of antibody using quantitative elisa.
How can i know the expected Kd value (range in nM, or mM)?
Relevant answer
Answer
If you do not have prior information on the affinity of your scFv, it's reasonable to start with a broad range that covers common antibody affinities. Based on typical affinities, a starting range of 0.1 nM to 1 µM should be adequate.
  • asked a question related to Antibodies
Question
3 answers
We are researching the conjugation of various antibodies to quantum dot microspheres and europium microspheres. The sustainability results have been unsatisfactory. Which stabilizing buffers do you recommend for stabilizing the conjugation step?
Best regards
Relevant answer
Answer
The appropriate stabilizing buffer for conjugating quantum dots (QDs) to antibodies is critical for ensuring the stability and functionality of both the QDs and the antibodies. The buffer utilized is determined by a number of factors, including the type of QDs, the conjugation chemistry, and the antibodies used. However, some general rules can help you choose an adequate buffer:
  1. pH: The buffer should maintain a pH that is compatible with both the quantum dots and the antibodies. Typically, a pH range of 7.0 to 8.5 is used for conjugation reactions.
  2. Buffer Components:Phosphate-buffered saline (PBS): Commonly used because it is biologically compatible and maintains pH stability. A typical concentration is 10 mM PBS with 150 mM NaCl. HEPES: Another buffer that is often used, especially for reactions that are sensitive to phosphate. It maintains a stable pH in the range of 7.2 to 7.5.
  3. Additives:Bovine Serum Albumin (BSA): Often added at concentrations of 0.1% to 1% to prevent nonspecific binding and to stabilize proteins. Tween 20 or Triton X-100: Low concentrations (e.g., 0.01% to 0.1%) of non-ionic detergents can help reduce nonspecific binding without significantly affecting the conjugation process. Glycerol: Can be added up to 10% to improve stability during storage.
  4. Chelators: Avoid using chelators like EDTA or EGTA if the conjugation involves metal-affinity interactions, as they can chelate metal ions essential for the quantum dot stability.
  5. Reducing Agents: Avoid using reducing agents such as DTT or β-mercaptoethanol during conjugation if the chemistry relies on disulfide bonds, as they can disrupt the bonds necessary for the conjugation.
Example Buffer Composition
A commonly used stabilizing buffer for conjugation might look like this:
  • 10 mM PBS, pH 7.4
  • 0.1% BSA
  • 0.05% Tween 20
Conjugation Chemistry Considerations
  • Carbodiimide Chemistry: Often used for conjugating carboxyl-functionalized QDs to amine groups on antibodies. Buffers like MES (2-(N-morpholino)ethanesulfonic acid) at pH 5.0 to 6.0 are commonly used for the activation step, followed by conjugation in PBS.
  • Maleimide Chemistry: Used for thiol groups on antibodies. Buffers should be free of primary amines and free thiols.
Final Tips
  • Buffer Exchange: Ensure that the antibodies and QDs are in the same buffer system before starting the conjugation to avoid precipitation or aggregation.
  • Optimization: Perform small-scale trials to optimize the buffer conditions for your specific QD and antibody combination.
  • By carefully selecting and tailoring the stabilizing buffer, you can successfully attach quantum dots to antibodies, assuring the stability and usefulness of the bioconjugates that result.
  • asked a question related to Antibodies
Question
2 answers
Our western blotting for tyrosine hydroxylase consistently shows double bands (50-70 kDa) in murine hearts using the #AB152 antibody from Sigma-Aldrich. Has anyone else encountered this issue? If so, could you please share your experiences with anti-TH antibodies that work well in cardiac tissue? Thank you.
Relevant answer
Answer
Thank you A.B Bayazid
  • asked a question related to Antibodies
Question
1 answer
Dear all,
have an antibody that stains the surfaces of my brain slices strongly, but the center is weakly stained. Please see the attached PDF for a comparison of three stainings (scanned in X-Z-mode). The bottom staining is the one that needs improvement.
Since we would like to quantify the staining in optical sections throughout the whole slice, I need to improve the labeling.
General protocol:
  • Mouse brain fixed in 4% formalin overnight
  • 50 µm thick vibratome-sections
  • Free-floating staining
  • No retrieval
  • Blocking for 60 Minutes in PBS with 5% NDS + 0,3%Tx-100
  • Primary AB 1:1000 in PBS + 3% NDS+0,3% Tx-100 over night at 4°C
  • Detection with Donkey anti Primary 1:500 in PBS + 3% NDS (60-120 Minutes incubation at RT)
Modifications I have tried (that did not solve the issue):
  • Longer incubation time
  • Antigen retrieval (citrate-based)
  • Dilution series from 1:250-1:64000
As you can see in the PDF, one can see this effect in staining 2, too. Both stainings 2&3 have in common, that the labeled protein is located in the membrane. I do not observe this issue in cytosolic proteins.
Unfortunately, my analysis requires a certain section thickness and the ability to analyze more than just the surface of the slice.
I would be grateful for ideas about how to fix this issue.
Best regards
Henrike
Edit for clarification: I replaced "intracellular proteins" by "cytosolic proteins" (PDF not updated). All three epitopes are located intracellularly.
Relevant answer
Answer
Hi,
One suggestion is that you reduce the section thickness. I normally freeze my sample organs in OCT and while sectioning, I don't go over 25 micron. You mentioned that you need a certain thickness, so I wonder if this suggestion would be applicable.
When you say longer incubation time, what is the length? Are you doing 48 hours or more? I have seen some papers where some weak antibodies are incubated for even 7 days.
  • asked a question related to Antibodies
Question
3 answers
I have FITC labeled proteins, and I would like to detect its fluorescence after running proteins western blot. Does fluorescence quench in WB condition? so I can detect FITC labeling (without using antibodies).
Relevant answer
Answer
The question is, how could you label the protein of interest among a mixture of proteins before purification?
  • asked a question related to Antibodies
Question
2 answers
Dear all,
I am in search for ideas for what might cause the following issue:
I have done IHC-IF extensively and wanted to perform stainings on cultured cells now, too. However, I am observing unspecific staining of all primary antibodies tested. It is a mystery to me!
Here are the details:
I am staining murine cell lines that have been fixed with 4% PFA for 10 Minutes.
Protocol (washes performed, but omitted here):
  • No retrieval
  • Blocking for 60 Minutes in PBS with 5% NDS + 0,3%Tx-100
  • Primary AB in PBS + 3% NDS+0,3% Tx-100 over night at 4°C
  • Detection with Donkey anti Primary 1:500 in PBS + 3% NDS (60 Minutes incubation at RT)
Results: Three different cell lines (neuronal, astrocytic, endothelial) all stain equally for the antibodies used (broad variety from different species), e.g. anti-GFAP, anti-GFP, anti-MAP2... Most antibodies tested work well in IHC (some are untested) and - based on the literature - many of them are used in ICC successfully. A control without primary antibody does not show staining, so autofluorescence or unspecific binding of the secondary antibody do not seem to be the issue. The composition of the media the cells were cultured in differ between the lines.
Steps taken so far:
- Used new buffers, aliquots, cells etc.
- I have done a dilution series with two different antibodies:
  • Antibody A should only stain cell Line A and antibody B should only stain cell line B.
  • Both cell lines were stained with both antibodies in 11 dilutions ranging from 1:500 to 1:512K
  • Result: Both stainings A and B do not differ between cell line A and B. One can see that the intensity is clearly reduced with lower concentrations of the primary antibody, but there is signal. This signal is absent in the control without primary antibody.
I have also planned to do a qPCR next week to verify the identity of the cell lines just to be sure, but I am feeling like it is a technical problem. I cannot come up with a reason that makes the specimen "sticky" for different primary antibodies, but not for secondaries. It seems unlikely to me that there is a problem with all primary antibodies tested, especially since they work well in free-floating slices.
Any ideas what might cause it and how to solve it would be very much appreciated!
Henrike
Relevant answer
Answer
Dear Yuning,
thank you for your suggestion! Unfortunately, the problem exists across all antibodies and cell lines tested. Some of these certainly should not react, e.g. anti-GFP in cell lines that do not express GFP. One could consider this a control similar to your suggestion - indicating that there is a more general issue which I cannot pinpoint.
Best regards
Henrike
  • asked a question related to Antibodies
Question
3 answers
I have an antibody specific just for ICC and WB but I should use the same antibody to perform an IHC staining. Is it possible to use this ab also in IHC?
Relevant answer
Answer