Tips on re-use of primary antibody in Western blot - (Mar/08/2009 )
Can you re-use secondary Abs-HRP @4C in TBS-T?
medchemgirl on Nov 19 2009, 12:16 PM said:
Long term, nope, HRP breaks down very fast in water. During 1-3 days probably, but activity will start to decline.
Reusing your secondary can help you to remove unspecific binding if you have some.
If you pretend to prepare a replenish stock or something like this, it should be in 1% PVA to stabilize de HRP.
The good thing about this is that such a high amount of PVA will give very clean background.
Uuuuuuu That sounds nice!!! Thank you!
Feelcontraire on Nov 19 2009, 07:41 PM said:
medchemgirl on Nov 19 2009, 12:16 PM said:
Long term, nope, HRP breaks down very fast in water. During 1-3 days probably, but activity will start to decline.
Reusing your secondary can help you to remove unspecific binding if you have some.
If you pretend to prepare a replenish stock or something like this, it should be in 1% PVA to stabilize de HRP.
The good thing about this is that such a high amount of PVA will give very clean background.
we've used primary antibodies over and over ( at least 5-6 times before noticeable loss in signal). we never store in milk or bsa and don't like using azide. we block our membranes first with milk or bsa...rinse 3X with 1XTTBS..then add the antibody diluted appropriately in 1XTTBS. after incubation we collect the antibody solution and filter sterilize it into a 50ml falcon tube and store at 4C. we then re-block the membrane briefly and proceed with secondary.
saves a lot of money.
antibodies are so expensive. usually, I will reuse the antibody within 1-2 weeks, have tried antibodies in 2% skim milk, three weeks old, 4 degree, it still worked but less 10 times sensitive as fresh one. after 1 month, it didn't work at all.
hope it helps