Analyze the Data 3-3: Protein Purification The purification of a microtubule mot

Question

Analyze the Data 3-3: Protein Purification

The purification of a microtubule motor protein has been undertaken (see Vale et al., 1985, Cell 42:39–50). A partially purified cell fraction containing the motor protein was subjected to an additional purification step using gel filtration chromatography. Aliquots of each fraction eluted from the gel filtration column were subjected to SDS gel electrophoresis. Fractions 21 to 41 are shown on the gel below, which was stained with Coomassie blue.

What differing properties of proteins likely account for the bands on the gel observed in lanes    

      21–26 versus those observed in lanes 27–36?

      This question is referring to the size of the proteins in the various fractions.   For reference,

      fraction 1 will elute first and fraction 41 would elute last from the gel filtration column.

Explanation / Answer

A 110 kDa protein is observed in SDS-PAGE for fractions 27-36. Further, there are doublet bands of 65 and 70 kDa observed in these lanes. These proteins are not observed in fractions 21-26. The gels are stained with Coomassie blue, which will stain all proteins eluted out. In the fractions 21-26, high molecular weight band above 205 kDa are observed.

Gel filtration chromatography separates proteins based on size and shape. The separation corresponds to the molecular weight. The gel filtration matrix consists of microscopic beads containing pores and internal channels. Large molecules will not pass through the beads and will be penetrate the beads. Larger molecules will flow around and in between the beads. Smaller molecules will be trapped in the pores in the bed. Hence, large molecules will eluted faster in the column than the smaller molecules.

Fractions 21-26 are eluted before fractions 27-26. Hence, fractions 21-26 will elute out the large molecular weight fractions. Fractions 27-36 elute the smaller or low molecular weight fractions. The concentration of the proteins in these fractions increase with fraction # as more proteins will be eluted.

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