I guess it depends on what you are looking for. If you don't want things denatured (i.e. what to preserve protein-protein interactions, then do NOT use Laemmli because it contains SDS, so it will just denature everything). But if you want a certain protein that is associated with a particular cellular compartment, LB may not be suitable, but a particular lysing buffer would be (i.e. Tris-Triton for cytoskeletal-bound cytoplasmic bound proteins).

Honestly, I usually lyse my cells directly on the plate with RIPA or NP-40, then take the protein concentration, then add LB to denature.

I recommend you read this very nice article by Abcam regarding lysis buffers.

http://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1

Hopefully some of that helps.

-b

I guess it depends on what you are looking for. If you don't want things denatured (i.e. what to preserve protein-protein interactions, then do NOT use Laemmli because it contains SDS, so it will just denature everything). But if you want a certain protein that is associated with a particular cellular compartment, LB may not be suitable, but a particular lysing buffer would be (i.e. Tris-Triton for cytoskeletal-bound cytoplasmic bound proteins).

Honestly, I usually lyse my cells directly on the plate with RIPA or NP-40, then take the protein concentration, then add LB to denature.

I recommend you read this very nice article by Abcam regarding lysis buffers.

http://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1

Hopefully some of that helps.

-b

Thank you very much for your thorough response. I will check the link.

Can anybody tell me full protocol for lysing samples directly in lammelli sample buffer? Why there are so many types of lysis buffers? How to choose which one to use?

I use the following recipe:

10% glycerol + 62.5 mM Tris pH 6.8 + 2% SDS

I warm this up to 95 degrees, add it to the plate, and scrape the cells after a minute or so.

HI Pallavi,

ideally a buffer is with tris or hepes with physiological pH at 7.4, concentration being 20-100mM with 100-150mM of Nacl (provide osmotic shock to cells duirng lysis); a detergent- choice of detergent depends on location of extracted protein- if membranous little stronger detergent, cytosolic - milder like NP40, if the protein of your interest is phoshorylated then use phosphatase inhibitors, protease inhibitors and a osmolyte like 10% glycerol taht acts as a cryoprotectant.

tnx. Thts true. But just the same protein by boiling in lammeli buffer which contains sds and tris, a protein lysate can be made without adding any protease inhibitors, then why lysis buffers with so many components an inhibitors are used are used?

Tnx, i think the link given above is very gud to read for my question. I have read it and i am convinced