Yesterday I reconstituted my antibody in 100 ul deionized water. Subsequently I diluted the antibody to 100 ug mL in PBS and aliquoted the stock. One aliquot I used to do my immuno assay in which I bound the antibody to a protein AG coating. t I stored the antibody dilution I used for my assay at overnigh at 4 degrees. The next day I tried to repeat this but the antibody did not bind at all. What could have gone wrong?
If very dilute protein is stored in some types of glass or plastic the protein may stick to the tube wall and if there was not much to strat with then there may be no free antibody left. Silanised glass or plastic vials may work better or adding some protein that binds to the tube and so blocks your antibody from binding. Albumin may work if it can be used without interfering with your assay
Hi Danny, in addition to what Paul said, and from my experience in ELISA, you have also to verify the pH of the all involved buffers including distilled water. The pH recommended range should be 6-7.
I think it is straight from the dilution. So diluted could mean the antibody could not bind at all to the sample. What has happened to the second vial?
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