For months, I have used F4/80 antibody on zinc fixed slides with a concentration of 1:1200, suddenly for the past two weeks, the 1:1200 dilution was not working and after reoptimizing it only 1:100 dilution is working well not even 1:500.
Has this happened to anyone before?
Any reason why this may have occurred?
The antibody is from the same company and yes different LOT number.
But does a different LOT number give such a drastic result?
Hello Shweta Johari
Yes, it is possible. Are you using polyclonal antibody? Polyclonal antibodies are raised against the full-length protein, against large protein fragments and against small peptides. In addition, there are differences between antiserum, protein A/G-purified, and antigen affinity purified polyclonal antibodies. Batch-to-batch variability is likely because these antibodies are produced in different animals at different times. When an antibody is raised to an entire protein, multiple parts of the protein will generate a collection of different specificities and affinities.
Also, most of the vendors do not manufacture all the antibodies. Most of their antibodies have been obtained from a wide variety of different manufacturers from all over the world. The vendors show that they themselves are the primary source of all their antibodies. This enables them to keep QC data on the product sheet that were generated many years ago thus keeping the sales going, while the actual antibody that generated these data may have sold out and has been replaced by successive other batches (from different animals), and the current batch on sale may no longer be able to generate such data at all.
Please refer to the article below for more information.
Article Commercial antibodies and their validation
Best.
it the F4/80 mab is directly conjugated to an enzyme and the tube was not shipped or handled properly, then there can be a dramatic drop in the performance of the antibody. Too hot or too cold will damage the enzyme when it is diluted down to working strength. You can report the problem to the company that you bought it from and request another lot.
Malcolm Nobre Respected Sir,
Thank you for your response. I hope this reply finds you well.
Sir, my antibody is not polyclonal but thank you for the article and the giving me an insight on the commercial production of Ab. That may be the issue.
Thank you again.
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