Usually there are several (even more than 10 or 20) antibodies for a protein in the market but how do you know which antibody is better or best for your experiment? Just try it one by one? Sometimes the company shows a good picture about the antibody at their website, but the antibody does not work......Does anybody have a good idea?
Many suppliers sell the same ABs lots, changing only the cat. no. according to company rules. If you have chosen one you want, check another for the price other sellers. On the other hand avoid the ABs you don't trust with similar description in the next company.
The negative control (KO mice, RNAi in cells, deleted gene in microorganisms) shown on the WB from the company page means you should buy it immediately. Overexpresed gene as an positive control is also nice, but not convincing.
Never buy the ABs if seller is not indicating what was the AA sequence of antigen used.
For WB the standard are polyclonal one raised against the epitope verified by BLAST for the presence in other proteins. But the old one monoclonal published before are the best, as many scientist in this discussion pointed out before me.
Every out of known companies sold me completely useless ABs, thus there is always a gambling when you are ordering. The trick I use to save the money is to buy a 1ml from Santa-Cruz in the price of 0.1ml from others. They do not recommend to freeze them I always do on the aliquot, never affected their activity. Then I have the ABs with the WB well set up I can do for many years (stored in aliquots -20). I don't know how they (S-C) are doing it but their ABs are dead at the experience date (1 year) when you store them at 4 degrees as they recomend...
Typically, I start with what other reputable researchers have published. If they have the same cell lines and protein X looks clean and bold, that is the one I order. Antibodies can vary from batch to batch so if company X sends you a below par antibody, call them back and they'll usually send you a new one.
Go through the respective literature and check, which antibodies have been successfully used by others. Usually, you will find some references on the company websites, which you can start with or do a Pubmed search for your protein of interest. It’s a tedious task but there is no way around it, at least in my opinion.
If you are lucky, some companies will be willing to give a very small aliquot to try for free. But I strongly agree with the previous replies. Go through the literature and find what others have used for similar applications.
I never follow the pictures of any companies but you know just after some time from experience that certain companies produce better ABs than others. For instance in my experience most santa cruz ABs are of inferior quality compared to Cell Signaling. The ones which in our hands also work better are some which have been raised against mouse or rabbit. When a AB only exists from one vendor and that as goat then I'm already very doubtful it will work well and very often that's also the case. These are things you can consider BUT mainly > publications and not only one as many as possible and it's important to look at their WBs to see if it's good or not and also at higher IF journals.
cell tech, abcam usually are stable. BD are good for flow. others, you might want to find publications. I have tried many santa cruz antibodies but majority of them are very dirty. for human genes, you can also check out the human protein atlas. they have validation on human paraffin sections (the antibodies are mostly from sigma and they do have a matrix for evaluating antibody specificity).
There is no guarantee, that specific antibody will work. Look for published clones, but there is still some variation between different batches. What usually gave us trouble where antibodies from Santa Cruz, while abcam, BD, Sigma worked fine. You will have to try it in the end (also the dilution) and make sure you use a positive control there.
Santa Cruz gets a bum rap, but many of our most useful antibodies come from them. Yes Cell Signaling is usually quality, but really they sell a lot of phospho antibodies that are cross-absorbed with non-phospho peptide so they always look 'clean'.
After initial identifications of our proteins with commercially available Abs, we use to routinely validate via HPLC. Relatively large quantities of those proteins are required however. It works very well for us but it is a very tedious approach with several purification steps required. We are shifting now towards characterization of proteins via an LC/MS/MS approach. The most recent advances with that type of technology allows detection in the low femtomole ranges (for example: http://www.absciex.com/products/mass-spectrometers/tripletof-systems/tripletof-5600-system-x51008).
Please note that I have no commercial interest with that company.
Check the epitope of the antibody. Select those which cover highest number of amino acides at interested part of your protein.
Before you purchase an antibody, make sure that the vendor indicates it is compatible with immunoblotting. Antibodies that work well for immunofluorescent staining may not be optimal for Westerns, because the epitope context is very different in the two methods.
Many suppliers sell the same ABs lots, changing only the cat. no. according to company rules. If you have chosen one you want, check another for the price other sellers. On the other hand avoid the ABs you don't trust with similar description in the next company.
The negative control (KO mice, RNAi in cells, deleted gene in microorganisms) shown on the WB from the company page means you should buy it immediately. Overexpresed gene as an positive control is also nice, but not convincing.
Never buy the ABs if seller is not indicating what was the AA sequence of antigen used.
For WB the standard are polyclonal one raised against the epitope verified by BLAST for the presence in other proteins. But the old one monoclonal published before are the best, as many scientist in this discussion pointed out before me.
Every out of known companies sold me completely useless ABs, thus there is always a gambling when you are ordering. The trick I use to save the money is to buy a 1ml from Santa-Cruz in the price of 0.1ml from others. They do not recommend to freeze them I always do on the aliquot, never affected their activity. Then I have the ABs with the WB well set up I can do for many years (stored in aliquots -20). I don't know how they (S-C) are doing it but their ABs are dead at the experience date (1 year) when you store them at 4 degrees as they recomend...
Prof. Pedro D'Orléans-Juste is right about the sensitivity of the detection of the M-S, is very suitable for pre-purified proteins. But still the ancient western I find as more "fashionable" for the proteins with: low expression level in crude tissue extracts; trypsin resistant; truncated forms you can miss in MS.
I agree with Prof Jan Piwowarski; good old westerns are still the gold standard for protein with low expression in crude extracts. Perhaps the first question would have been to ask which type of proteins are targeted here. Trypsin digestion can be a problem indeed with some proteins. Approaches are however available to try to circumvent that particular problem (please refer to: http://cdn.intechopen.com/pdfs/29629/InTech-A_critical_review_of_trypsin_digestion_ for_lc_ms_based_proteomics.pdf ) .
Interestingly, in the last decade, several papers have reported extensive LC/MS/MS proteomic profiles from blood vessels, among other tissues. The proteomic profiling is generally perform by 'blasting' the MS data in protein databanks (several of them are available online and are 'compatible' with data generated from LC/MS/MS).
Cell signaling and Abcam have always worked well for us. And they have always allowed us to try another lot or refunded our money on the very rare occasion when our antibody did not work. Also signaling-gateway has a nice resource of antibody ratings....
http://www.signaling-gateway.org/data/antibody/cgi-bin/targets.cgi
check what's published! you can ways check the literature and see what antibodies people use and how their results (and protocols/conditions look like).
the first hints you can find in antibody databases like:
http://www.antibodypedia.com/
http://www.proteinatlas.org/
What others successfully used should also work for you :)
There are some prerequisites:
1. The epitope should be stable even when the protein is SDS-treated / denatured, therefore a linear epitope. You can check this by an spot assay (spot the SDS-treated antigen(mixture) on a NC-membrane in decreasing concentrations [1 µL]. Incubate this with decreasing concentrations of your potential antibodies. use this checkerboard titration to find the antibody with the lowest detection limit ... and the highest affinity respectively. Those high affinity antibodies show normally only a small degree of cross- or multireactivity)
2. The antibody should fit into your reaction scheme. Beware of the species differences. Your labeled antibodies should not cross react with antigens in your sample. This becomes more important if you would go for double or triple staining by using different antibodies at the same time on the same western blot strip.
3. The antigen-specific antibody should not cross react with other antigens in your sample. In most cases it's helpful if an antibody works in immunohistology and Western blot. In the histology you have a bunch of antigens present at the same time.
You want to make sure that the antibody was created against either a very pure protein, a peptide or cDNA. other than that, it is key that it is a linear epitope, not a conformational epitope, and thus is must be demonstrated for use in SDS-PAGE or Western.
It's really surprising how much your blocking buffer will affect Ab performance. You can go from no bands (or lots of spurious bands) to a beautiful blot by changing the blocking buffer. It may be worth a quick optimization experiment to try a few blockers and see if a different blocker can rescue your "bad" Ab. I've attached a link to a paper on this topic.
http://www.ncbi.nlm.nih.gov/pubmed/18563731
To address your question, in this paper
http://omicsonline.org/2155-9872/2155-9872-4-168.pdf
There is described method for comparison of antibodies from various Ab providers. It is called Dot-ELISA affinity test. If you succeed in obtaining an antibody testing aliquots from various companies, you can test and compare them by this assay and choose the most suitable for you
Dear All, I am beginner in WB for Fasciola hepatica/ Fasciola gigantica please inform me about latest trends in their proteomics or ABs
Here you go: https://dewan-pavel.blogspot.kr/2017/05/tips-for-choosing-best-antibody.html
I'd like to echo on what many have stated here: Go through literature search to find which antibody has been published in a experimental context similar to yours.
BenchSci (www.benchsci.com) is a free resource for all academics that curates published data based on your specifications (ie. Western blot for ERK1/2 in rat brain tissue).
I hope this helps!
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