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[proteo]

Has anyone used protein free blocking buffers before? I know Pierce sells one that is suppose to eliminate high background. My blots have high uniform background after I tried blocking in a non-animal protein blocker from Gilead Biosciences.

[Feelcontraire]

proteo, on Nov 18 2009, 10:04 AM, said:

Has anyone used protein free blocking buffers before? I know Pierce sells one that is suppose to eliminate high background. My blots have high uniform background after I tried blocking in a non-animal protein blocker from Gilead Biosciences.

You can use PVA(polyvinyl alcohol) (size 140-180k).

The original article talks about 1 part per million for few seconds.
I usually prepare a 100ml container with higher concentrations which works better, and ad thimerosal 0,1% to avoid fungus grow. I change it from time to time.
To use it I usually live the membrane there 1 minute.

If you increase the concentration you will get even better non-background.

The original article uses a different molecular weight(maybe 50-100k) and they find that it works as a standard block, ie more block less background and less signal.
But in a patent about HRP preservation I read that the PVA of molecular weight 130-180 doesn't seem to interact with interact with proteins, thus blocks membrane but not protein interaction.

I bought mine from sigma but it will certainly work from any other source, just make sure that the molecular weight is on that range.
I usually make a 2% stock, it is not easy to dissolve if you don't follow the instructions:
-Heat water to 60-70ºC
-Remove from heat and with agitation slowly pour the PVA dust.
-It may take some time to disolve, use agitation, and heat (never exceed 80ºC)
-Don't heat from the bottom with the PVA in as it may stick to that hot bottom.
-Heat will readily help it to dissolve, you can for example microwave for time to time.
-When it is disolved ad thimerosal 0,05% to avoid fungal grow.

10 parts per million is what I use (100ml water 50ul of my 2%stock and 1ml of 10% stock of thimerosal)
As much as 1% can be used.

Edited by Feelcontraire, 18 November 2009 - 11:53 AM.