Suitable lysis buffer to extract transmembrane protein? (eg. tyrosinase) - (Jan/16/2008 )
Hi, I plan to lyse B16F10 mouse skin melanoma cells (in 96-well plate) to release the tyrosinase transmembrane protein using Triton-X. What is the suitable phosphate buffer for the preparation of this lysis buffer?
Can I use 0.1 M sodium phosphate buffer (pH6.8) containing 0.1 % triton-X? I should store the lysis buffer in the fridge (4 degree celsius) or at RT?
Do I need to add in protease inhibitor such as PMSF?
Have the buffer at 4C before adding it to the cells. 0.1% triton should help. If not, you might have to use higher conc. of triton or stronger detergent.
Have some EDTA in the buffer and add either some protease inhibitor (We use a protease cocktail from sigma) or PMSF.
Use the best buffer system that you will use later on to carry out the reaction, supplement it with 0.1% triton X-100, protease inhibitors, DTT, EDTA, as scolix suggested. 4 C for storage.
Have some EDTA in the buffer and add either some protease inhibitor (We use a protease cocktail from sigma) or PMSF.
How much concentration of EDTA and PMSF/protease cocktail I should prepare in 0.1 M sodium phoaphate buffer (pH6.8)?
Have some EDTA in the buffer and add either some protease inhibitor (We use a protease cocktail from sigma) or PMSF.
How much concentration of EDTA and PMSF/protease cocktail I should prepare in 0.1 M sodium phoaphate buffer (pH6.8)?
1-2 mM is fine for EDTA, be sure that your enzyme does not require Ca2+, Mg 2+ for its activity, or you have to add an extra Ca2+, Mg2+ to the reation system. In one of our protocol, we use 25 mM PMSF final conc. For cocktails, you just add one pill to 50 ml lysis buffer. The kit has the info.
Have some EDTA in the buffer and add either some protease inhibitor (We use a protease cocktail from sigma) or PMSF.
How much concentration of EDTA and PMSF/protease cocktail I should prepare in 0.1 M sodium phoaphate buffer (pH6.8)?
1-2 mM is fine for EDTA, be sure that your enzyme does not require Ca2+, Mg 2+ for its activity, or you have to add an extra Ca2+, Mg2+ to the reation system. In one of our protocol, we use 25 mM PMSF final conc. For cocktails, you just add one pill to 50 ml lysis buffer. The kit has the info.
the protease inhibitor cocktails are in tablet form?
I use Roche's, yes, it is.
Yeh, they are in tablet form, but we have one thats in liquid form (sigma).
Yeh, they are in tablet form, but we have one thats in liquid form (sigma).
Which type of protease inhibitor cocktail should be used for protein extraction from animal cell line? Use the one for general use (lyophilized powder form) or the one specific for mamamlian cell lysis (DMSO solution form)?
Comparing PMSF and protease inhibitor cocktail, which one is easier to prepare and suitable to be used?