Protein kinase protocols - (Feb/16/2011 )
Hello everyone.
I wanted to post this so that people that experiment with proteases could help. I'm trying to standardize a protocol for probing protein kinase C from cell lysates from QC and GM-95 cell lines. The protocol that we are using is the following...
Lysate buffer
20 mM Tris HCl
0,5 mM EDTA
0,5 mM EGTA
0,3 mM Sucrose
0,5 mM PMSF
We expose the cells (grown in 50 ml bottles) to a toxin and wash it two times with PBS and for each time we lysate the cell culture adding a cockatil buffer and lysate buffer to each sample. We proceed to homogenize with a tuberculin syringe. Then a 15 minutes centrifugation at 8000 rpm at 4°C. Then we separate the supernatant from the pellet that will correspond to the nucleus fraction.
Then we centrifuge at 100000g for 1 hr at 4°C, and we separate the supernatant and the pellet. We add to the pellet 80 ul of lysis buffer with 1% of triton x-100.
We then proceed with the quantification which sometimes is really scarce of protein.
I'm wondering if anyone that works with protein kinase could help me find a better way to do my experiment.
you will need a lot (and I think more) of cell material to run this protocol which is o.k.
Do you take the whole triton soluble fraction? And what about the triton-insoluble fraction?
anacris28 on Wed Feb 16 20:23:03 2011 said:
Hello everyone.
I wanted to post this so that people that experiment with proteases could help. I'm trying to standardize a protocol for probing protein kinase C from cell lysates from QC and GM-95 cell lines. The protocol that we are using is the following...
Lysate buffer
20 mM Tris HCl
0,5 mM EDTA
0,5 mM EGTA
0,3 mM Sucrose
0,5 mM PMSF
We expose the cells (grown in 50 ml bottles) to a toxin and wash it two times with PBS and for each time we lysate the cell culture adding a cockatil buffer and lysate buffer to each sample. We proceed to homogenize with a tuberculin syringe. Then a 15 minutes centrifugation at 8000 rpm at 4°C. Then we separate the supernatant from the pellet that will correspond to the nucleus fraction.
Then we centrifuge at 100000g for 1 hr at 4°C, and we separate the supernatant and the pellet. We add to the pellet 80 ul of lysis buffer with 1% of triton x-100.
We then proceed with the quantification which sometimes is really scarce of protein.
I'm wondering if anyone that works with protein kinase could help me find a better way to do my experiment.
We take the whole triton soluble fraction that will be 80 ul and for the insoluble triton we had it separated in the 100000 g centrifugation with a volume of 500 ul. The problem that we have been facing is that the protocol is not so reproducible and we sometimes are unalbe to observed the activation of protein kinase C which is our main purpose.
Inmost sun on Fri Feb 18 10:33:38 2011 said:
you will need a lot (and I think more) of cell material to run this protocol which is o.k.
Do you take the whole triton soluble fraction? And what about the triton-insoluble fraction?