1- 3% BSA blocking ?
2- Primary antibody
3- HRP conjugated secondary antibody
4- Transfer buffer
5- Running buffer
Hi there,
I would say they can all be reuse except HRPconjugated secondary antibody for which I doubt about enzyme stability in time (I always use it freshly diluted though I actually never tried to reuse it). For 1 and 2, addition of azide 0.01% avoids bacterial growth for a few days if solutions are kept at 4°C, the other thing to think of is actually the number of probing done with primary antibody solution within the period; if number is high antibody depletion may occur and also the volume tends to decrease. For transfer and running buffer, you can actually recycle them until you notice the running and transfer efficiencies are affected.
Blocking buffer: Stable for several days, but cheap enough that it's often worth making fresh. Could be made more stable with preservatives such as azide or thimerosal. I often re-cycle my blocking buffer as the diluent for my 2' AB -- in which case adding a preservaitve like azide would be a bad ides (may inhibit the HRP).
Diluted Primary Antibody: Stable for months if preserved with azide or thimerosal and stored at 4*C or cooler (some people freeze diluted ABs, I do not).
Diluted HRP 2': Stable for several days, but cheap enough that it's often worth making fresh. I've used the same aliquot of diluted 2' AB for several blots when performing serial blotting, but I wouln'd trust it to remain active for more than a few days in solution.
Transfer Buffer: Can be re-used many times. I've not kept track of my many times I've been able to use a batch of transfer buffer, but it's north of 10x Transfers. The big issue is that the buffer begins heating up faster and faster during the transfer as the buffer ages, requiring more rapid replacement of ice packs or more aggressive means of cooling.There's probably some chemical changes going on in the buffer, but I'm honestly not aware of what they are. By the time the buffer starts overheating too rapidly I usually just make fresh buffer.
Running Buffer: I'm not sure if it's reusable, but I suspect it is. I usually make it fresh every time.
BSA for 4 hrs. Primary and secondary antibodies for 1.5 hrs. Wash buffers for 10 minutes and 3 repeats. They are suitable times for very good results.
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