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Size exclusion column chromatography was used to separate the proteins in a Drosophila cell lysate to attempt to identify the protein complex responsible for processing long dsRNA into siRNAs. SDS-PAGE followed by silver staining (Figure 1A) was used to analyze the fractions collected, and the fractions were tested in parallel for their ability to cleave long dsRNA into siRNAs. The dsRNAs were separated on a denaturing polyacrylamide gel (Figure 1B).

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the siRNAs have nt=21

The question asks: Which fractions are enriched for siRNA cleavage activity?

My thinking is that the fractions 19,20 and 21 are enriched for siRNA cleavage activity due to the more intense blobs at nt21 in Fig.1B...Is this correct?

Also, how can I estimate the molecular weights of the protein(s) in the complex that is likely to be responsible for the dsRNA cleavage activity?

my thinking is that the two bands present in all three lanes are the ~190kDa and ~33kDa proteins which should be responsible for dsRNA cleavage. The others do not seem necessary...

Please could someone correct me in the right direction by offering their reasoning? Thank you!

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  • $\begingroup$ Sounds about right though you need to explain how the results in B help you to determine A. You should use band intensities comparisons for each of the lanes to make your case. Remember that the ability to rule something out is just as important and the ability to determine something, so explain why you rule out what you rule out. $\endgroup$ – AMR Jan 4 '16 at 3:01

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