I have been running many western blots and have not been able to always use fresh primary antibodies each use because of a limited supply. The proteins were isolated from cells treated with different drug does (including a mock treatment). After normalization to beta-actin, the fold changes of the target proteins relative to mock apparently had too much variability. One hypothesis is that the old age or reuse of the primary antibodies is causing the problem. This doesn't make sense to me yet because the antibody should be mixed well in its buffer and have plenty of time shaking overnight at 4degC to fully bind to their target. Will this cause any variability once the data is normalized to the loading control and then to mock? Old and reused antibodies may be more dilute and will therefore show weaker bands, but the fold changes between those band should still be equal.
Another possibility is reuse of the blocking buffer, but I still don't get how another change consistent within each blot would affect the fold changes derived from each blot. Is there some enzyme kinetics or densitometry concept that I am missing, or could the variability be coming from another source? Thanks.
Hi Luke,
I perfectly agree with you, antibody reuse cannot create that bias you observe and should only result in a reduced blotting efficiency (faint bands) at best. I think that the explanation should be searched elsewhere. Have you run your mock/treated samples on the same gels? Have you extracted and quantified mock/treated proteins on the same day from the same experiment? I am afraid that the only thing I can suggest, if you didn't do already so, is to quantify again your protein extracts and re-load a new gel. Probably is just a normalization bias if I understood correctly, should go away with a more careful protein quantification. Surely not the ab reuse.
Hope it helps
Good luck
Francesco
Hi Luke, if you are using a monoclonal Ab for detection, then there could be 2 problems here. 1. loss of the epitope with age. 2. loss of monoclonal Ab. with age.
Dear Luke,
what you said is theoretically right, but then, the reality is more complex. If I right understand your problem is that : you have the same samples, (lysates of cells ,treated with different drugs ,that should change the amount of the protein of your interest) loaded on different gels but the intensity of the signal (relative to the mock) is different when you use old or reuse antibody.
first of all, I don't understand if these samples are the same or they are biological duplicate. if they are biological duplicates, obviously, you can't exclude that the problem could be relative to the treatment.
Second suggestion is to try a new normalizer and to check fold change with the new one ( GAPDH, or tubular, or what you have in lab).
third: did you do ponceau? is ponceau in line with your normalizer? because, it could be possible that the bands of your normalizer, that should be an antibody that work in a very good way, are saturated and then when you do the fold change, you loose the difference.
moreover, you shouldn't grant the fact that an antibody in its mother buffer will be always ok. it could be possible that some one let the antibody at room temperature for error for a couple of hours, and then your antibody doesn't work finer as previous; I can say to you, for personal experience, that I became crazy, with an antibody that works only for a couple of weeks from its shipment. we used the antibody in every single combination and then speaking with others we discovered that it was a common problem with that antibody.
however my last suggestion is to take a membrane that give to you a strange result, re-ibridizes with the fresh antibody, and if you obtain your expected result, order big amount of antibody, since you are working with an antibody that can't be safely reused.
regards
Francesco
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