Reuse primary antibody - (Feb/11/2006 )

Hi, everyone!

I would like to know if is possible to reuse the primary antibody , and how many times .
thanks

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depends on the company...i use cell signaling ones 3-4 times within a span of 15-20 days. Even if the antibody has been used just once and is more than 20 days old i chuck it.

With santacruz (everyones fav punching bag) i use them 2 times within 5 days and if i use 50 ul in 10 ml of milk then the second time i add another 10 ul to the same and reuse. But SC is no good than 2 times....

and i donot add sodium azide as a preservative as i remember fred_33 saying that it was really hazardous so...

another thing to be considered is the kind of antibody...if it is phospho-something i use it just twice even from CS...if it is total-smothing 3-4 times

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hi
i use eurogentec and santacruz antibodies for p53 or TBP for the most of my westerns. 6uses of p53 shows no reduction of detection, whereas secondaryantibody is able to be used 4times (but after decreasing o efficiency).

For tough westerns, 2uses at most.
i don't add anything in my ab, but in general, do the first hybridation o/N at 4° and second at RT. after that, o freeze at -20°C.
The milk don't smell bad after 6uses...

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I thought i was the only one that subjected my antibody dilutions to the "smell test"

good to know someones out there doin the same...

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it's the simpliest test i know.... It's also the first question i ask to a student that came to me and says my western didn't give me results...

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