小中大
CRITICAL STEP The maximal protein load can be limited by large amounts of neutral detergent in the sample and by high
concentrations of lipid when solubilizing biological membranes. SDS must always be in large excess over neutral detergents
and/or lipids. For isolated mitochondrial membranes (70% protein, 30% lipid), for example, the optimized maximal protein load
of sample wells 0.7 × 5 mm is ~20 μg. Increasing the applied amount of protein to
40 μg, for example, may cause the individual protein bands to disappear in a diffuse
background. Around 0.4% neutral detergent in the sample can be tolerated for
direct mixing with SDS-containing sample incubation buffers and application to SDS
gels. The incubation buffers and volumes described in Step 4 set a tenfold excess of
SDS over the neutral detergent. Setting the SDS/neutral detergent ratio to <10 may
result in a surprising result after staining: normally separated, large proteins may
be detected in the upper gel areas, but the lower gel areas may be completely clear
with no small proteins detectable.
是不是这个问题？
