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
Hello fellow researchers,
I'm having a puzzling problem in my Western blot experiment, can somebody help me? I conducted an experiment using prefrontal cortex samples, following a WB protocol that has previously yielded successful results (We took quite some time to standardize each step). However, this time around, I'm facing a situation where I'm not able to detect any bands, despite thoroughly checking various aspects of my protocol.
Here are some key details:
- My samples were homogeneized in RIPA buffer + proteases inhibitors as usual, and are relatively fresh, I homogenezeid last month, and I am realizing western blot with those samples since that.
- I run my electrophoresis in BioRad system, at 150V, 400mA, 2h, room temperature (10% acrylamide gel)
- I transfered to nitrocelulose membranes in semy dry transfer 30V, 1h, 164mA, room temperature
- I performed a Ponceau staining and confirmed that the samples were transferred correctly to the membrane (image is attached)
- I used three different antibodies in those membranes in the first time (I cut the membranes in three different sizes), it didn't work and I thought that it could be a old antibody solution problem. So I stripped the membranes and I incubated with new antibodies solutions (I got three new and sealed antibodies, including the secondaries) and none of them resulted in detectable bands.
- I was very careful to see that I incubated the correct primary antibodies, with their respective secondary antibodies
- Blocking (BSA 5%) and washing steps (3x with TBS-T) have been successful in previous experiments with those antibodies of the same brand.
- The protein quantity in the samples appears adequate, as good bands were visible in the Ponceau staining.
- I'm using high-quality and well-maintained Super-ECL reagent.
I'm completely stumped by this situation, especially because even the internal control protein, beta-actin, is not being detected. If anyone has faced a similar issue or has suggestions on what else I can investigate, please share your insights. Any assistance or guidance would be greatly appreciated.
Thank you for your attention and help!
Just curious if there is so much endogenous GST within cells, why would you use a GST-tagged recombinant antibody and surely you will purify/detect interfering endogenous enzyme when using this system? The sequence of the GST Tag and the GST (pi isoform) found in most cancer cells is 80% similar according to a BLAST of the sequences. Any light anyone could shed on this would be much appreciated. Thank you
I want to model antibody structure but I do not have the sequence of the whole antibody. I just have the sequence of its CDR region. Do you know any web server to model antibody structure by grafting CDR regions on the structure of the antibody?
I am currently planning an experiment that involves viewing E. coli cells tagged with gold-conjugated secondary antibodies using a scanning electron microscope, and I am running into the issue of cost for primary antibodies. I might have the option of using primary antibodies previously purchased for Western blots, but I am unsure if these antibodies can also be used for SEM imaging. I do not yet know enough about the chemistry and reactivity of antibodies to answer this question, thus I find myself here!
On a related note, if anyone has any recommendations of good websites to purchase primary antibodies for E. coli that work with SEM, I would love some! I have found a few websites, but each of them only has 2 or 3 antibodies for this purpose.
I've recently transitioned to a new institution, and I have limited experience with flow cytometry. In my previous work with microscopy and western blots, blocking with proteins was a standard practice to prevent unspecific antibody binding. Here, however, flow cytometry is conducted in PBS without any added protein throughout the protocol. I'm perplexed because I've always believed that the addition of protein is essential. Could you please help me understand if using just PBS is sufficient to obtain specific results? Thanks!
We have bought a detection antibody and used it to run an indirect ELISA. Initially, we were not getting meaningful readings when diluting the recombinant standard sample to the picogram level, so we changed the dilution concentration of the recombinant protein to the nanogram level and got readings.
Should I just get the capture antibody in the matched pair and switch to doing a sandwich ELISA instead and follow the manufacturer's recommended standard dilutions of 1000 pg/ml - 8 pg/ml? Scouring through the literature, everybody uses kits and not manual making of ELISA, even less people use indirect ELISA as a method. So I am here asking for help and advice, any papers appreciated!
Hi everyone! I am new in lab and I have been having problems with Western Blot, I use a Chemidoc and when I reveal I see nothing, after reincubate, or incubating with a new antibody, the signal is lost, or, is very very low, when I dye with red Ponceau, I see a lot of protein because I put 40 ug per lane, I don't have idea about what happened, someone could help me, I will be eternally grateful
I am trying to develop my western blot using an antibody targeting a 17kda protein. It is binding non-specifically and not giving any band(like in the photo). What does it mean is happening? How should i trouble shoot this?
Our lab has always ordered the goat anti mouse highly cross preadsorbed secondary antibodies from Thermo but we find that there is a lot of batch to batch variation in terms of cross reactivity with rat. This is a problem for us as one of our labs favorite antibodies is a rat tubulin antibody that we like to combine with mouse antibodies. While a work around has been to first incubate mouse primary, then mouse secondary, then the other primary antibodies followed by their secondary antibodies, it would be preferable to find a reliable source of mouse secondaries that do not show cross reactivity with rat. Any recommendations?
Does anyone have a recommendation for a good phospho-parkin (Ser65) antibody to use for western blot? The cell signaling one didn't really work for me...
I am encountering bacterial growth in my diluted western primary antibodies (in TBS, without any milk/bsa, with 0.01% NaZ). We keep the antibodies in +4 C since we use them frequently (Our incubations are also o/n at +4 C). Almost every 2-3 weeks I observe the contamination. I filter the antibodies with 0.4um filter every 1-2 months.
I am wondering why there is that much of bacterial growth even with NaZ. Also, is there a better way of decontaminating antibodies? Can I keep the antibodies in -20 C (how many times I can freeze/thaw them?)
Does anyone know of a good antibody for immunohistochemistry to stain for DARPP-32?
Or does anyone know of an immunohistochemistry antibody to stain for D1 receptors?
Or an alternative suggestion for a protein to stain as a marker to indicate a D1 agonist injection
Hello, I am interested in determining whether the binding of our antibody to a cell membrane receptor causes the receptor to be internalized or not. We have data that suggests the antibody prevents receptor cleavage, but this could be due to the antibody preventing proper cleavage or simply causing internalization.
My initial idea was to conjugate our antibody with a fluorescent label (Fluorescein), incubate the antibody with our cells (3T3s), wash the cells to remove unbound antibody, and image the live cells immediately using confocal microscopy to see if the conjugated antibody was internalized.
I have limited imaging experience with fixed cells, but would this idea work? Should we consider doing live cell imaging instead? If so, would the process (incubate, wash, image) be the same?
I would appreciate any help.
We've been looking at counting amyloid plaques in mouse hippocampus using 20x fluorescence images (antibody D5452). However when using either ImageJ or Matlab code, the fill holes or watershed options have thus far not worked to account for these dense core areas or partitioning plaques. I was wondering if anyone had further suggestions?
I would like to find out the amino acid sequence for a few CD3 antibodies. Does anyone also know any platform/database that I can find amino acid sequence of antibodies?
I need to convert the fg/ml, and ng/ml limit of detection into IU/ml. What is the formula for converting fg/ml, ng/ml limit of detection into IU/ml?
In mouse SI-lamina propria T lymphocytes stimulation with PMA (20ng/mL) /Iono(1ug/mL) is not strongly inducing IFN-g after 6hrs. The previous results in our lab had 4-5% and I could have just 2% max, though the protocol and antibodies to stain everything is followed in same way! any suggestions or comments would be greatly appreciated.
Bref A (1ug/mL) after 2hrs of PMA7Iono.
I would like to know if the binding between extracellular vesicles (EVs) in serum and immunoglobulins is strong or not.
Our Antibody Tissue Culture department has had issues with Myco contamination in the past. We are currently having to dissassemble our Clarifying and Concentrator TFF units to soak the Millipore filters and concentrators every weekend. The idea is to avoid risk of myco contamination of filters/concentrators.
However, disassembling the units this often is an ergonomic concern.
My question is: Is there a better way to handle this issue?
My suggestion to the team is that we allow the cleaning buffer to run through the filter in reverse and forward motion for a longer period of time, as opposed to letting them soak over the course of two days. I consider the motion to be efficient in cleaning the unit thoroughly.
We are purifying custom-made antibodies from frozen chicken serum, and when thawed it turned into the gelatin-like substance on ice. So, their erythrocytes are nucleated and then I treated serum with DNAse, thinking that it is a genomic DNA released, it helped a little, I centrifuged, took out super, put on ice and it turned into the gelatin again. It can't be loaded on the column like that. What is it in their blood and how to get rid of it? Thank you!
How do I determine the appropriate sample size for a study focused on developing a new immunohistochemistry antibody to detect a breast cancer biomarker and assessing its performance compared to a conventional antibody for the same biomarker?
It is known that Antisperm antibodies are produces in cervical fluid and serum of married women because of the antigenic ilicit caused by spem transported to the female genital tract during intercourse .
the question light on the causes behind detectin of ASA in unmarried women?
I am conducting research on OT-1 murine extracted CD8T cells and their activation and proliferation. Which method is more effective for stimulating their proliferation: CD3/CD28 antibody or OVA peptide? If CD3/CD28 antibody is preferred, what are the specific steps involved? Alternatively, if OVA peptide is suitable, what is the detailed protocol for inducing cell proliferation using this approach? Any additional tips, considerations, or references related to the activation and proliferation of OT-1 murine extracted CD8T cells would be highly appreciated.
Thank you for your valuable insights.
Hello, first I shall share that I do not have a lot of familiarity with this topic so excuse my ignorance if there is any.
We know that anti-drug antibodies exist, with some against small molecule drugs, usually these are haptens bound to some proteins.
At some level of structural complexity, it becomes difficult to produce an antibody that is selective enough to target the specific antigen without off target effects.
My question is this.
1) What is the simplest structure which antibodies can be formed against? i.e. what is found experimentally?
2) Is there some sort of theoretically imposed lower bound on the informational complexity of an antigen against which antibodies can form.
I should mention that I am aware of riboswitches and their ability to recognize individual ions and discriminate them from other ions, even with identical charge.
I am using 100 and 120 nm latex particles and monoclonal and poly-clonal anti-crp antibody. i tried with Tris, Glycien, PBS, and MES buffer with different molarity (10 to 100 mmol) but not getting linearity more then 100 mg/L.
I saw some other people presenting similar problems.
For a few years now, we were using Dynabeads M280, coupling them to a biotinylated antibody to perform immunopurification of target molecules. All of a sudden, after opening a new lot of magnetic beads, the beads do not seem to work anymore, and all of the biotinylated antibody can be detected in the unbound fraction by indirect ELISA.
We did not change anything in the protocol and checked the pH of all buffers already. The problem presents itself with all the different biotinylated antibodies that we checked, all of which are from the same lot or even from the same aliquotes that previously worked well.
We contacted the company, and tried other beads lots, as well as the magnetic beads from a competitor.
The way I see it, the binding step between biotin and streptavidin is clearly the problem, and we tried different conditions (time, temperature, rotation vs agitation) in order to improve it, but nothing seems to work anymore.
I was wondering if someone is still using such beads successfully, or has ever encountered any problems of this kind.
Thanks in advance!
I did the subcellular fractionation of transfected HEK-293T cells.
For western blot analysis for the purity of fractions, I used anti-B actin antibody, anti-fibrillarin G4 antibody and anti-calnexin antibody for cytoplasmic, nuclear and membrane fractions, respectively.
Anti-B actin gave expression both in cytoplasmic and nuclear fraction. Later, I realized that B actin is also present in the nucleus!
With Anti-Fibrillarin, expression was detected in nuclear fraction only.
However, with Anti-Calnexin antibody, expression was both in nuclear and membrane fraction, with higher amounts in the former.
I was wondering, if that could be possibly be due to the contamination of nuclear fraction with membrane fraction.
Could anyone suggest me more specific markers for identifying the three fractions?
Thanks in advance.
For any new vaccine antigen, it is inoculated to target species and the blood is collected to assess the antibody titre. And in a particular time, the subject is challenged with the live organism. Now my question is how to determine the protective titre?
I need to run western blot for total and phospho form of a protein. I am planning to transfer the proteins to the membrane, probe for the phospho form first, strip and re-probe for the total protein next. Is this method correct? Will there be some phospho antibody remaining in the membrane after stripping which will interfere with the binding of the total protein antibody later?
For the last 2 years, I have been producing antibodies in 293 Freestyle cells for my PhD project, and tested them against cancer cells, by verfying, first of all, binding to such cells. However, since the beggining of this year, the produced antibodies stopped binding cells. In charaterization by western blot, besides the band I would normaly expect, I see now an extra band with increased molecular weight. I tested the whole procedure, from production to purification, and I don't think the problem is in there. So my question is: is there any chance that the cells would change something in their machinery that would impact translation, folding ou post-translational modification on the antibodies, ou production of any other product? I have been using a relatively low passagem from the initial commercial stock (P.7). Would it be plausible? Any other tips?
I attached a picture of a blot with intense nonspecific staining at the bottom of the nitrocellulose blot. The left blot, from left to right, shows a ladder, a 20ug load and a 40 ug load. The blots were incubated in different primary antibodies; Left blot was Rabbit anti-human Taz and right blot was Mouse anti-human GAPDH. Both used the same secondary antibody, polyclonal goat anti-rabbit Alexa 647 (Thermo fisher # A32733) and were photographed with a BioRad ChemiDoc MP Imaging system. Antibody incubations and blocking were conducted using ThermoFisher Superblock in TBS, 20% in TBST. The right blot effectively served as a negative control b/c the goat anti-rabbit did not recognize the mouse primary antibody.
There is a very bright stain near the dye front at the bottom, seen in both the left and right blots. Why this is so intense? Would it be worthwhile to cut off the bottom of the blot before staining to reduce this stain? Any other suggestions? Thanks for your help.
I am training to stain by Immunofluorescence for Tau protein on mouse cortical neurons, I used Monoclonal mouse Tau antibody( ref: 314011synaptic system) with dilution 1/1000 and also I tried with another Monoclonal mouse Tau antibody (ref:MAB3420Sigma-Aldrich) with dilution 1/1000 as well. For both antibodies I tried with different fixation method, Methanol fixation for 5 mins at-20 ºC/Methanol-EGTA(1mM) for 5 mins at -20ºC/Methanol 5 mins at-20 ºC followed by 4%PFA-4%Sucrose at room temperature and 4%PFA-4%Sucrose at room temperature. My neurons are DIV14 but with these fixation methods the 2 different antibody are not working except with methanol-PFA fixation but I do not think it is specific since I think i should be only positive in axon but I still see the staining every where
So, I wonder if some one have already use these antibodies and manage to have a good staining?
Any recommendation would be appreciated, Thank you in advance
These antibodies will be used to validate some antibodies by IHC. Thanks
I shake microglia (200rpm, 2h) from P0-P3 mice after 12-14d culture of primary mixed glia. After another 3-4 days culture, I stain the cells with Iba1/GFAP/Oligo2 antibody, but I find that all these markers can stain every cell. Has anyone encountered this? What is the possible reason?
My IF protocol: 1. wash three times with PBS, 2. fix cells with 4%PFA and 120nM surcose in PBS for 15min at RT, 3. 3 x 5min wash in PBS, 4. block cells with 3% donkey serum, 5. incubate cells with 1 antibody over night at 4℃, 6. 3 x 5min wash in PBS, 7. incubate cells in 2 antibody for 1h at RT, 8. 3 x 5min wash in PBS.
I have conducted western blot experiments with rat brain tissue, where I have tried to measure the GAT-1 concentration. I am using the antiSLC6A1 antibody from Elabscience (Catalogue Number: E-AB-66423). Similar to the example in their website, I have detected GAT-1 blots closer to 55 kDa. However, when I look at other antibodies for GAT-1, I see that the blots are closer to 70 kDa (which is a lot closer to the mentioned molecular weight of GAT-1 in antibody manuals). Has anyone got similar results and somehow worked their way around?
I have a tube of antibodies marked by the manufacturer that can stain frozen sections and paraffin sections for immunofluorescence, but the manufacturer did not indicate whether it can stain immunofluorescence of cells. I wonder if such antibodies can also be used to stain cells for immunofluorescence. Has anyone encountered a similar problem, looking for your answer.
I need to produce a custom antibody and I need the company to also synthesize the peptide. Does anybody have experience with a company that they liked?
I am working to increase the dynamic range of a sandwich ELISA and the robustness. I have worked on the capture antibody concentration, primary and secondary antibodies concentration, the plastic, the blocking, coating and washing buffers. The robustness is now OK by changing the buffers but the dynamic range stay still very low. Have you got any ideas?
My aim is to identify and amplify the variable regions of mouse Ig and to further clone the sequences in a suitable expression system.
I'll be working on conjugaison reactions with monoclonal antibodies, have somebody already done that before ? This is my first time using monoclonal antibodies ? What about excipients ? Should I purify the antibody before using it ?
It would be great if someone could enlight me on this.
My best regards
I am doing the PC12-C6 co-culture system and would like to double stain the cells to observe the cell to cell interactions. I first used the mouse FITC-conjugated anti-beta III tubulin antibody, and then used propidium iodide to stain the cell nuclei. Will the signals from propidium iodide strong enough to eliminate the signals from anti-beta III tubulin antibody?
Activation of naive T cells generally requires antigen to be presented by dendritic cells in lymph nodes. Activation of naive B cells generally requires opsonized antigen to be displayed by follicular dendritic cells in lymph nodes.
Thus, it seems that dendritic cells and complement (innate immune system) need to recognize a pathogen before an adaptive immune response can be initiated. Is this always the case?
we are currently having trouble with our Westernblots.
Attached you can find two pictures of our blots.
We use fluorescently labelled antibodies, but even if we do not incubate the blots after transfer with the ABs, we see this problem. So it seems so be some kind of autofluorecence.
Changing the buffers, using different water and so on, nothing really help. Sometimes, the blots look ok and sometimes these red clouds occur, without any relation...
Does anybody have an idea what to do or change to get rid of this?
Thanks in advance.
I have read protocols for development of antibodies from rabbits post immunization with peptide. However, I shall be the first to do this in our lab. Can anyone suggest how to perform (video/tutorial) the rabbit immunization step by step with precautions?
I did a western blot with 25ug of protein lysate. As control, i used the recombinant protein and cell who not express my protein. The first antibody (rabbit polyclonal) recognize only the recombinant form. A second antibody (rabbit polyclonal) recognize the recombinant and the endogenous forms. Why does the first antibody not bind the endogenous form ?
I have tried to check the purity using Abcam's membrane fraction antibody (ab140365). If you have used the same antibody, can you please share your blots?
I am working on exosome characterization by identifying the CD markers through Western blotting. Recently in one of the experiment I have come across a strange problem in the transferred PVDF blot. After the immediate transfer of the bands from gel to membrane, I have seen a definite band at 50kda. But after probing with Antibody of my interest, the that band which is at 50kda length is also my protein of interest, has disappeared during chemidoc imaging.
Could anyone plz help me with this?
Moderna claims that detection of Covid19 Anti-Spike IgG antibody found in the lungs of Rhesus Macaques (that have high natural immunity against the virus) was evidence that the Spike Protein antibodies resulting from jabs in the arm circulated in Blood and somehow penetrated the Blood-Lung Barrier.
However there is an interesting paper from very early in 2019 that shows Anti-Spike IgG causes Severe Acute Lung Injury by skewing macrophage responses.
A doctor named Joseph Lee has challenged Dr Fauci, the editor of New England Journal of Medicine and many other scientists, plus the mRNA jab manufacturers to prove that the insides of lungs are protected by their LNP delivered materials.
He reports that they don't give satisfactory replies to his questions about the Blood Lung Barrier and has offered a reward for any proof via Twitter.
His website carries useful details.
Could Alveolar Macrophages be a conduit for antibodies?
I purchased the human clone of this gene and sub cloned it into a Xenopus oocyte plasmid (it contains Xenopus oocyte beta globin to enhance the expression in Xenopus oocyte) with a polyA site. But no functional activity of this protein was detected (I repeated the experiment 3 times). I sequenced the clone and it is correct.
From the literature, I know that its rodent and fish homologues, and some of its family members in human were successfully expressed in Xenopus oocyte with detectable activities without expressing their known co-factor(s). I found an unpublished dissertation work online saying that they couldn't detect function of this human clone in Xenopus oocyte either using a different functional assay.
I ordered antibody of this protein hoping to see whether it didn't express or its function was inhibited for some reason. While waiting for the antibody (it will take weeks), is there anything that I can do to help figure out the reason why I didn't detect its function?
I'm trying to stimulate human T cells with magnetic beads coated with antibodies. I have streptavidin-coated beads as well as biotinylated antibodies. According to the vendor, they usually incubate cells with antibodies first and then mix them with beads. I'm wondering if it's feasible to immobilize the antibodies onto beads first and then add to cells. Thanks!
Hello! We used plasmid pNeae2 to display nanobodies on the surface of E. coli DH10B-T1 R using the bacterial display methodology. Then we attempted to use this bacterium as a primary antibody in an indirect ELISA assay to test its affinity with a protein. If anyone has any suggestions regarding this methodology or references related to it, please let us know.
tags: nanobodies, single domain antibodies, VHH antibodies, indirect ELISA assay
Has anyone used an Nkx2.5 antibody for immunohistochemistry with nuclear signal? The sc-8697 antibody works perfectly, but it has been discontinued and we are struggling to find a replacement. We have tried other commercial brands or the monoclonal alternative, but all of them provide an unclear/fuzzy staining.
Any recommendations are welcome!
I am doing an experiment that requires me to fix some microbial cells, stain with antibodies, run through the flow cytometer and ultimately isolate DNA. The whole procedure is roughly 6 hours. Usually I fix cells for flow with PFA, but I'm concerned this may interfere with the DNA extraction and/or lower the DNA integrity. Can anyone suggest a more suitable fixative, something that wouldn't take more than 1hr?
I used mouse antibody for b-actin and got multiple bands. other proteins (dll4/jag1) did not show multiple bands. alpha-tubulin looks better but has some broken bands as well.
I need to inject an antibody into the mouse's brain and record synaptic currents from brain slices 6 days after the injection. Is there any innocuous fluorescent molecule I could include in the injection solution that shows me in the slice where the antibody was placed? Thank you!
I have an antibody that I want to buffer exchange into PBS, as the current buffer contains high concentrations of a carrier protein. I do not know the concentration of the antibody, but that the antibody is at a low concentration (~50-100ug/mL). After buffer exchange via desalting column, I used Nanodrop to determine the A280 of the resulting material, and received a concentration of 2 mg/mL. This is much higher than the predicted concentration of the antibody. Is it possible that the buffer exchange was incomplete and needs to be performed again?
The protein was extracted from mouse tissue(brain)，marker is Thermo 26619.
The ladder was about in 25kd.And the antibody is BD Bioscience#asyn antibody.
I'm not sure if it was α-synuclein.
We are getting results that are too similar to each other on our UV-VIS spectra for very different molecules. We are in need of a procedure or help with building the appropriate way of going about this characterization. We are working with fullerenol, sulfo-SMCC, and an antibody. The buffer of this solution is PBS-EDTA as well as some DI water. What should our baseline be? is water fine? Should we use all PBS?
As for the reference, is a clear cuvette appropriate or should we do PBS in the reference as well?
I'm working on nanobodies that incorporate propargyl-lysin (PrK) in its sequence. I observed that most of my protein was in dimer form prior to loading it to the antibody column, and that in this form, the nanobody was unable to bind to it and was lost. The dimers aren't formed by disulfide bonds, and I don't know what kind of interactions the PrK might have that would cause them. I would appreciate any assistance from anyone who has experienced such dimers with PrK.
We produce this MAb by culturing the hybridoma line in CL1000 flasks using DMEM (the specific formulation provided by ATCC) supplemented with 20% FBS & antibiotics. We purify the antibody by rProtein A affinity chromatography (eluted using 0.1 M sodium citrate buffer at pH 5.5). The eluted antibody is then dialyzed against PBS pH 7.4 and the sample becomes turbid. The ELISA titer of the pre dialyzed material is far better than that post dialysis. Any suggestions will be greatly appreciated.
I have the following question: I have a nested PCR protocol that uses a chemically modified hot-start polymerase. However, in the lab, I have a hot-start polymerase, but bounded to an antibody. The purpose of the PCR reaction is to amplify a specific fragment of a viral genome, which will subsequently be sequenced by the Sanger method. The question is, can I use the polymerase I have, and will the antibody that blocks the polymerase activity before the appropriate temperature, interfere with the subsequent sequencing analysis? Thank you for your answers!
Can anyone recommend me some good antibodies for western blotting/ flow cytometry targeting human P2X7R (P2X7)?