The protocol refers to a stock solution. Perhaps the 10X stock solution belongs to a kit.. But I have used casein as a blocking agent at final concentrations in the range 1-4% (w/v). That means 1-4 g od casein in 100 ml of the buffer. Casein is vey difficult to solubilize.You can do it with a magnetic stirrer. If casein is slowly added to the buffer while stirring you can solubilize it. Alternatively skimmed milk contains a lot of casein and is easily solubilized. You can use milk as your blocking agent instead with excellent results.
Thank you very much for your reply, I'll try your suggestion and let you know about the results.
regards
Houshang
We adapted this protocol in which casein ins resuspended in an alkaline solution and then the neutralization and addition of NaCl results in a casein solution in TBS. Also included is the tech note that we probably used as a starting point.
The key is to neutralize with HCl very gradually. You will have to be patient or the HCl will coagulate the casein and you will have cheese in your blocking buffer. Although we made the solution only every 3 months or so, a good investment was a buret with a stopcock so the HCl could go in drop by drop over a long period without supervision. Our protocol filters the solution at the end, but too much cheese and your casein concentration will have decreased. Good luck!
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