western blot samples trouble - (Jun/13/2005 )
i´m using a cell lysis buffer with triton and tris-HCL PH 7.5. i would want to make a western blot by i don´t know if i will have problems with the samples PH (stacking is 6.8) and with the samples migration, my buffer doesn´t have SDS!!Should i mix my samples with a buffer with sds and a appropiate ph?help me, please.thank you
First off, I am wondering why you are lysing cells in the first place.
Why not sonicate and spin to collect the pns?
Run the lowery for protein concentration and then take the volume of protein you need, suspend it in 2x Lammli, add DTT and go.
That way you don't need to worry about pH compatability.
An alternative to that is TCA ppt.
I use RIPA buffer (buffer is 50 mM Tris, pH 7.4, 1% Triton-X100, 1% Na-deoxycholate, 1% NP-40, 0.1% SDS, and protease inhibitor) to lyse cells and use microplate BCA assay (2 ul of sample in 300 ul of total volume) to estimate protein concentration. Then I adjust the sample volume with RIPA buffer to give me 4 ug/ul. Finally, I add equal amount of 2x SDS sample buffer (Molecular cloning recepie). I have no problem.
i supossed that i had lo lysate the cells because i´m trying to detect calpains (they are in a very low concentration in the citosol of cells, also they can be bind to membranes). First of all, i used a lysis buffer with sds, tris hcl 6.8, proteases inhibitors and pmsf, i used a bca assay to quantificate but i couldn´t detect the protein, so i´m trying to use another buffer with triton (more agressive) and after this i will sonicate the samples.do you think i could detect my proteins?thank you!
Triton is not more agressive than SDS. Triton X-100 is non-ionic surfactant, but SDS is strong ionic surfactant.
I can't promise you it'll work or not, but Triton X-100 sure would help since it "can" extract only cytosolic fraction (this is the main reason why I use RIPA buffer). Perhaps contribution of nuclear protein is insignificant in your case, but at least you are getting rid of them to increase the concentraton of cytosolic protein. If it still won't work, the only option is to lyse more cells.
thanks vagrants, i´ll try!!!!!mmmm.....another question, i´m working with human chondrocytes, do you know how long should i sonicate ??
IF you are going to lyse the cells on the petri dish with Triton, don't bother to sonicate... both are methods to break open the cells.
If you want to scrape your cells off the plate (lets assume a 100mm dish) add 10ml PBS and spin them down 10 min 2700 rpm in swinging bucket rotor. Then resuspend in 500 ul of buffer and look at the sample... it should be cloudy or turbid with cells. Sonicate for about 10 seconds... the sample should now look opalescent. Thats how you know they are sonicated.