The method is the same as you would use for SDS-PAGE or IEF-SDS-PAGE. The quality of the final Western image is going to depend on what you put into the gel. So the normal factors apply. Make sure the gel is overloaded, the salt concentration is low (for IEF it needs to be ~zero) and that contaminants (lipids, DNA etc) have been removed. For a lysate, the amount and the dynamic concentration range of the sample loaded will be the biggest issue for getting good resolution in the gel, which will be then transferred to the membrane.
You can use ready to use RIPA buffer or you may prepare it by yourself
Abdelfatteh Can you guide me which recipe of RIPA is best to use as there probably 3 on the basis of Ph 6.8, 7.4 & 8.0 then there is a phosphate based & tris based buffer ..!
Are you using mammalian cells? if it is the case I will send you the recipe of RIPA buffer and depending on your purpose to explore phosphorylated protein and total protein expression. Could you send me more details/
I am using mammalian cells (mouse). The protein of my interest is certainly phosphorylated as well , please send me the recipe (Email : [email protected])
Usually I use Tris-HCl pH 7.4, and before I proceed to the lysis I add protease inhibitor cocktail from Sigma to RIPA buffer.
Suspended cells and adherent cells the protocol is little bit different.
Obviously not sonication.
See the buffer reported by Alhamdani mohamed
Dear Rana
The are several methods to prepare samples for SDS-PAGE and also Western blot:
1. Grinding/homogenization: by using mortar and pestle, blender or tissue hemogenizer.
2. French press
3. Enzymatic lysis
4. Sonication
5. Osmotic shock
6. Freeze-thaw
7. Using of detergents
I usually use mortar and pestle with liquid nitrogen and work on ice to prepare cell lysate and avoid destroying of protein by protease. Due to possible cross reactivity of Igs in WB, you cannot use of protease inhibitors in certain buffers.
Cheers
Dear Masood, which buffers cannot be used with protease inhibitor ?
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