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Student Analysis Sheets

In protein separation by SDS polyacrylamide gel electrophoresis, migration is determined not by intrinsic electrical charge of polypeptides but by molecular weight. Sodium dodecylsulfate (SDS) is an anionic detergent that denatures proteins and confers a negative charge to the polypeptide in proportion to its length. Treatment of proteins with SDS and a reducing agent such as B-mercaptoethanol (BME), causes the individual polypeptides to become negatively charged with equal charge per unit length. By this means, we can determine the molecular weights of polypeptides. This is done by running a gel with standard proteins of known molecular weight along with unknown polypeptides. A linear relationship exists if the logarithm of the molecular weights of standard proteins are plotted against their respective relative mobilities (Rf). To determine the relative mobility (Rf) of a protein, divide its migration distance from the top of the gel to the center of the protein band by the migration distance of the tracking dye from the top of the gel.

distance of protein migration
Rf =
distance of tracking dye migration

A standard curve is constructed by plotting the Rf values of the standard polypeptides against the logarithm of their molecular weight. The Rf values of the unknown polypeptides is determined in the same way. The log of its molecular weight is read directly from the standard curve; the antilog is the molecular weight.


Experimental Results:

Experiment Analysis

(1) Compute the log of each marker fragment presented in Table 1. (Table T1 has been completed.)
(2) Measure the distance (in mm) from the top of the gel to each fragment. Try to be consistent: measure from the top of the gel to the middle of the fragment band. Record each in the Table 1, in order, beginning with the band closest to the top of the gel.
(3) Measure the distance from a sample well to the end of the gel.
(4) Calculate the Rf of each fragment. Record the data in Table 1. NOTE: The distances from individual wells to the end of the gel may not the same. Therefore, the Rf's should be calculated based on distances measured from individual lanes.
(5) On a sheet of graph paper, construct a standard curve plot using the molecular weight protein marker electrophoretic data. Plot Rf (horizontal axis) against the logarithm of each molecular weight in Daltons (vertical axis).
(6) From your standard curve (Figure T2) you can determine the molecular weight of the unknown polypeptides.
Note: The gel is 75 mm long. Therefore Rf values are calculated as migration distances (in mm's) divided by 75 mm.


Teacher's Notes:

The experimental data presented in the table is taken from the gel photograph in Figure T1. Individual student results may vary from these published results. Student data should approximate this data; the overall relationships of band placement should be consistent with the data presented in Table T1.

Standard Curve

Table T1
Data Table
High Molecular Weight Protein Markers
Protein Molecular
Weight
( Daltons)
Log Distance
Migrated
(mm)
Rf
Fragment 1 Urease 120,000 5.08 13 .18
Fragment 2 Fructose-6-P-Kinase 83,000 4.92 19 .27
Fragment 3 Catalase, Bovine Liver 60,000 4.78 24 .34
Fragment 4 Bovine Serum Albumin 66,000 4.82 30 .43
Fragment 5 Egg Albumin 45,000 4.65 39 .55
Fragment 6 Carbonic Anhydrase 29,000 4.46 50 .71

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