Solublising Casein for use as blocking agent - (Jul/03/2002 )
hi. A number of groups and protocols recommend the use of casein as a blocking reagent in elisa, western, and so on. I am aware that it is quite insoluble in water, but the question is, should i try to solublise it, or use it as an insoluble suspension? If i should solublise it, the big question is, how? I have read that it is highly soluble in alkali, but will this then be suitable for the applicationsa i have in mind? Hope you can help,
I use alkalisoluble casein (Merck) for blocking agent in western blot ( QIAGEN protocol).
casein preparation :
- make TBS + 1% casein pH 7,5 as :
1) tris 10mM + Na Cl 150mM + casein without adjust pH (pH 10) with stirring.
2) Adjust pH with HCl 1N at pH 7,5
3) stir at least 30mn and adjust the final volume
I have used 0.5% casein in 1xPBS as blocking agent in western blot and ELISA before, but I found it alright even the casein is not soluble. Just stir it for a while and then pipette the supernatant only