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. 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.

Molecular motors are ATPases that use energy of hydrolysis of ATP to generate a motive

      (movement) force. Hydrolysis of ATP results in the production of ADP and inorganic

      phosphate, the latter of which can be detected by chromogenic methods. How would you

      determine which fraction(s) obtained from both of the columns shown above contain(s) the

      molecular motor?

Explanation / Answer

Fraction number 28, 29, 30 ,31, 32, 39, 40 ,41, 42 and 43 -     are showing the bands for alpha and beta subunits(one and two) of molecular motor which generally ranges from 117 - 51 kDa. For the reference - Nadim Cortas, Deborah Elstein, David Markowitz and lsidore S. Edelman "Anomalous mobilities of Na,K-ATPase a subunit isoforms in SDS-PAGE: identification by N-terminal sequencing" Biochimica et Biophydca Acta, 1070 (1991) 223-228 Intensity of the bands and Molecular weights of the subunits may vary with the source, incubation time and temperature given for the sample preperation.

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