Can you answer this ASAP? Biotinylated TERRA transcripts were incubated with nuc

Question

Can you answer this ASAP? Biotinylated TERRA transcripts were incubated with nuclear protein lysates from primary MEFs and their association with the proteins hnRNP A1, A2B1, F, M, D/AUF1 and HuR was detected by western blotting. A biotinylated control RNA (C) corresponding to a random sequence of the same length as the biotinylated TERRA (N_48) was used as well. The RBP vigilin was used to monitor the evenness of sample input, as it was identified in both TERRA and control samples. Quantification of the TERRA signal/input ratio was carried out to assess the amount of endogenous RBPs bound to TERRA (see % bound to TERRA). Explain why the authors mean by the value assigned to % bound to TERRA After close analysis of the RBPs above list the TERRA binding proteins in order of greatest affinity to lowest affinity. Explain why we could not see these results in a coomassie stained SDS-PAGE gel

Explanation / Answer

Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97). CBB is the most popular protein stain. It is an anionic dye, which non-specifically binds to proteins. The structure of CBB is predominantly non-polar, and it is usually used in methanolic solution acidified with acetic acid. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.

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