Very easy question regarding biochemistry lab technique! 5. (3pts) Chromatograph
ID: 62446 • Letter: V
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Very easy question regarding biochemistry lab technique!
5. (3pts) Chromatography columns have a limited protein binding capacity During lab #4 you should not have ''overloaded'' the Ni^+2 Agarose column. Yet you may have noticed that the W2 fraction fluoresced slightly. If you see a band in the Western Blot W2 lane, what does this data say about the physical protein structure of rGFP in that lane? Give a possible MW for this band based upon the data (show your work) 6. (4pts) If you did ''overloaded'' your Ni^+2 Agarose column with a GCE sample that contained too much rGFP, would you expect to see a band in the W2 lane of the Western Blot? If so what is its MW? Would you expect to see a band in the E2 lane? If so what is its MW? 7. (3pts) you develop the Western Blot, what MW size band in lane E3 are you expecting to observe? If you happen to see two bands - one at the expected MW and one lower band - what might you specifically conclude about the physical protein structure of the lower band?Explanation / Answer
5. If the protein is seen in W2 lane, it suggests that protein has not bound to the Ni+2 column and physical nature of protein may be the histidine sites might have been buried inside the protein, which makes the protein unable to bind. The MW of the protein might same as that of the native protein.
6. Very less amount of protein would be seen in the W2 lane due to the overloading of the sample. In the E2 lane, more amount of protein can be seen as all the protein bound to the column will be eluted. The MW of protein in W2 and E2 lane would be same as both proteins are same.
7. The MW of the protein in E3 lane would be same as the MW of the protein in the E2 lane. If you are able to see a lower band, then you can conclude either some of the native protein is degraded or the protein may be a dimer having two subunits.
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