I have noticed a strange behaviour of the last two batches I have bourght of Qiagen´s anti-penta-HIS antibody.

After diluting it with buffer (1xTBS, 2% BSA, 0.2% NP40) 1:2000 it is fine for the first western. But when frozen and thawn again for reuse (which was not a problem for many other AB so far), it drastically looses activity and proteins are hardly visible/recognized on the western. At about the 3rd re-thaw event, it does not recognize proteins anymore at all.

The incubations are carried out in the cold room for about 1h. So I can rule out denaturation/degradation due to too high temperature.

Also I have tested different batches of buffer with no difference.

Has anybody also noticed this with the same or any other ABs? Can it be a specific problem of the AB that does not tolerate freeze/thaw-cycles (which is not said in the manual) or might it be due to the buffer (which is well tolerated by other AB)?

It gets pretty expensive by the time to use the primary AB only once.... :-(

is it possible that the antibody is being depleted by use? the antibody concentration may be lower than you expect and it may be getting nearly used up with the first blot.

you can try reusing without freezing to see if this is the case.

Hi,

some antibodies do not like freezing, and I'm afraid this one is one of them. We keep this antibody in the fridge, for long periods of time, and that way it keeps stable well. Try keeping the diluted one with some azide at 4C instead of freezing.

Cheers,

Erica