Apparent molecular weight determination experiment

Summary

Source: Laboratory Guide to Proteins and Proteomics

Operation method

basic program

Materials and Instruments

Target Proteins
Standard molecular weight proteins
Gel plate Electrophoresis apparatus Staining solution

Move

① Perform SDS-PAGE by upsampling the target protein and standard molecular weight protein onto the same SDS-PAGE plate gel.

Electrophoresis of target protein and standard molecular weight protein on the same plate gel eliminates differences due to different acrylamide concentrations and electrophoretic conditions. For finer molecular weight determination by this method, it is desirable to use acrylamide gels of different concentrations (Ferguson, 1964; Neville, 1971). A detailed discussion of the effects of both polyacrylamide and cross-linker concentrations on protein migration when using a variety of commercially available standard proteins for SDS-PAGE is given in Makowski and Ramsby (1997).

(ii) After electrophoresis, protein staining was performed.

③ (Optional or not) Dry the gel before measuring the ruler F value.

④ Measure the distance migrated by the protein and the tracer dye (usually bromophenol blue).

⑤ Calculate the relative mobility (Rf value):

RF = distance migrated by protein / distance migrated by tracer dye

⑥ Plot the half-logarithm of the known Mr using the mobility. The points in the middle of the gel should be almost in a straight line (see Figure 2.7).

(vii) Read the Mr of the unknown protein from the graph by the F-only value of the unknown protein.

Point: The standard curve should be re-plotted for each gel.

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