LiCl Cell wall wash
LiCl Cell wall wash
Do different cell types contain different wall assocation proteins?
1- Pellet cells (NC64a, SAG) 4 min at 4.5K in F14-6x250y rotor.
2- Suspend in a total of 8 ml pH 7 MOPS buffer
3- Take 4x2 ml aliquots of each (screw cap conical tubes)
4- To one aliquot of each add 220 ml 10M LiCl
4a- Save the other aliquots (cell suspensions) in the frig for further expts
5- Incubate 15 min at RT, then pellet cells (3min at 2K in S4180 rotor).
6- Save supernatants (cell wall wash), ppt 1ml aliquots of each w/TCA.
7- Dissolve pellets in 60 microliters borate cracking buffer w/2mM IAm.
8- 100C for 4 min, then run 3, 8 microliter aliquots on SDS gels