Hello Sir/ Madam,
i have prepared the bacterial outer membrane proteins by ultra centrifugation method
and stored in phosphate buffer saline(pH7.6) -20*c.I have started doing SDS PAGE of these samples according to Sambrook's protocol. 5%stacking gel (pH6.8), 12% resolving gel(pH8.8), tris glysin buffer (pH-8.3), 2x sample buffer (50mM tris, 2%SDS, 0.1% BPB, B-MERCAPTO , 10% glycerol).
I am running gel at 100mV const. voltage. by doing all described above i have been facing folowing promlems.
1. the major problem is,max. fraction of loaded protein struck either in stacking gel or in resolving gel and I could not get proper band separation. important point to take into cosideration is that SDS PAGE RULAR gave very clear protein band separation in same gel.
SMEARING AND SMILING ARTIFACTS ALSO TAKES PLACE
if any body have solution to my problem then please help me.