Keratin protein - Protocol for lysis (Aug/09/2005 )
Hi friends !
Anybody working with keratin protein? I want to find a way to lyse keratin. Though there are several protocols for keratin extraction but these are designed for tissues or structures which are available in plenty. I want to know of a protocol where I can dissolve the small amount of keratin which is present in my sample. Coz only complete lysis will give me distinct bands.
(I am doing One dimensional SDS-PAGE and I have tried conventional and Shindai method )
I use SDS lysis buffer for my keratins to go in sollution,
(Tris HCl pH 7.2, EGTA 5mM, EDTA 5mM, SDS 0.4% with protease inhibitor cocktail from calbiochem which is optional)
I assume that you have isolated keratins and then you want them to go in sollution. If this is not the case then there is a paper from Achstatter et al a very old paper of keratin isolation you can reffer it to. I am sorry if the spelling is wrong. If U are looking for lysing the cells and then do the keratin extraction then above buffer is good enoughf to sollubilize keratins.
If it does not work then you can sollubilise your protein in 8M urea where all the keratins are known to get dissolve.
Also some questions, you want to do amount of keratin in solluble and insolluble form ? then there are detergents and assays measuring that.
What exactly do you want to do?
I hope it helps.