Electrophoresis and Protein Separation (5C) Practice Test

A lab tested the effect of voltage on band resolution in SDS-PAGE (10% gel) for two proteins: A (50 kDa) and B (55 kDa). Samples were denatured with SDS and run in identical buffer at either 80 V for 60 min or 200 V for 24 min (same approximate run length). Band separation (distance between band centers) and band broadening were measured.

Results:

• 80 V: separation 0.35 cm; average band width 0.18 cm
• 200 V: separation 0.30 cm; average band width 0.34 cm

Based on the data, which factor most likely reduced separation efficiency at 200 V?

A researcher ran native PAGE at pH 6.5 to separate two proteins of different size and pI under a constant 140 V for 22 min. Wells were near the cathode (−). Protein M: 30 kDa, pI 6.0. Protein N: 90 kDa, pI 8.5. Migration distances toward the anode were M = 1.9 cm and N = 0.0 cm.

Which conclusion about the proteins’ net charges at pH 6.5 is most supported?