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
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!
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
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
Does anybody know of any functional anti-CD89 antibody or other reagent to block human IgA from binding to CD89 on cells? Thank you.
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.
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
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
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.
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.
Does anyone have a good antibody for Fsp1 (S100A4) that can be used for IHC on frozen sections (mouse tissue)? Thanks so much!
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:
- Which ASO conjugates (e.g., GalNAc, peptides, antibodies, polymers, etc.) have shown the highest level of target specificity in current research?
- Are there any conjugates that are proving to be more versatile and applicable across multiple targets or tissues?
- 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.
Does anyone have a good antibody for Fsp1 (S100A4) that can be used for IHC on frozen sections? Thanks so much!
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.
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?
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.
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.
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.
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!
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
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?
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.
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!
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!
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.
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.
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!
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.
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.
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?
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?
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.
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
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
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?
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
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?
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?
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.
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,
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
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)
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.
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.
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.
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
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?
I am trying to couple several antibodies (including antiCD14 and antiCD3) to magnetic nanobeads, after coupling they immediately form aggregates. Does anyone know why?
I have this problem with my western blot for the ABCG2 antibody on nitrocellulose membrane. Help please
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.
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?
I need to do immunohistochemistry in mouse brain, particularly in neurotensin-cre mouse line.
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?
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.
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.
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?
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!
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.
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?
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?
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?
Can anyone recommend high-quality GSDMD antibodies for immunofluorescent or immunohistochemical staining in mice?
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
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.
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)?
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
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.
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.
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).
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
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?