buffer for expression analysis - (May/24/2006 )

I have to check my protein expression either its soluble form or insoulble form. I have expression and i check its expression through SDS page and sample prepared by 5 X Tris Glycine Buffer and 95 C for 5 minutes but i want to check with out detergent. with out SDS.
Can any body tell me some simple buffer to test its expression in soluble form.
Freeze Thawing is lubrious process and previously i did but i get nothing not even other cellular
proteins. Please suggest me some simple buffer use.
I have an idea about phosphate buffer which is compose of 1 M K2HPO4 and 1M KH2PO4
and with lysosyme 2 ul for 1 ml culture and incubate it at 37 c for 1 hr. is it ok or any simple method to use.
with best regards
from
samita

We use triton extraction buffer to get the soluble proteins and study their activity.
here is the recipe.

TBS-Triton extraction buffer
50 mM Tris ph 7.5
150 mM NaCl
0.1% Triton X-100
10mM NaF
Add DTT to 1.5mM and PI to 1X immediately before extraction

Good Luck !!!

what is the protocol for extraction of protein. and what is P1

I apologise that I didnt include the whole protocol.

everything has to be cold, on ice.

Tris-Triton (extraction buffer) buffer + DTT + PI (protease inhibitor)

First remove medium and wash with PBS 1x
Add 300µl of the extraction buffer (300µl for single well in 6 well plate)
Rinse the wells with the buffer a couple of times
Collect the extract and then sonicate it briefly
Spin full speed – 5-10 min.

Supernatant contains the soluble proteins.

Collect supernatant and store in –80 C

Good Luck