protein preparation for Western Blot - (Sep/28/2006 )

Hi,

I am new with Western Blotting. After lysis of cells with RIPA buffer, what buffer/solution should be used to dilute the proteins for concentration determination or later use?

Thanks!

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Just add more RIPA plus protease inhibitors.

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I agree with rkay447.
We use protease inhibitors from Roche (Complete Mini Tablets) and they work very well.

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to determine the concentration, you should reduce as much as possible some components that can interfere with the assay, like detergent, reducing agent, salts.
that's why I recommend you to dilute a small aliquot in water, for the protein concentration determination. (and prepare the standard in diluted lysis buffer)

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If you can afford it, use RC DC Protein Assay from Bio-Rad which is specifically designed for samples containing detergents, reducing agents and more. You have a list of the non-interfering substances with the assay (in the package insert). Otherwise I agree with Missele.

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important is that blank controls are performed in the same solution as your proteins; dilution in water reduces side effects of lysis buffer components and detergents on concentration measurement

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hey. it's best to dilute your protein samples in dist water. as said before - the lysis buffer contain a lot of chemicals that interfere with your protein determination method. you can check the acceptable concentrations of these chemicals in any data sheet of your protein method. zee:)

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