8. (4pts) The oligomerization of Influenza A Virus matrix 1 (M1) protein was analyzed using affinity chromatography followed by gel filtration. (A) Purified recombinant M1 (with a C-terminal His6-tag) from nickel affinity chromatography eluted in peak 4. The fraction containing peak 4 (from panel A) was collected. SDS-PAGE analysis determined that the peak 4 fraction was 100% pure. (B) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 7.4 (physiological pH). (C) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 5.0.

The oligomerization of Influenza A Virus matrix 1 (M1) protein was analyzed using affinity chromatography followed by gel filtration.(A) Purified recombinant M1 (with a C-terminal His6-tag) from nickel affinity chromatography eluted in peak 4.The fraction containing peak 4 (from panel A) was collected.SDS-PAGE analysis determined that the peak 4 fraction was 100% pure.(B) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 7.4 (physiological pH).(C) Half of the collected fraction was applied to a Superdex 200 HR 10/30 gel filtration column at pH 5.0. Figure adapted from: Zhang K, Wang Z, Liu X, Yin C, et al. (2012). PLoS ONE 7(5): e37786. doi:10.1371/journal.pone.0037786 Which peak in panel B contains protein(s) with the highest molecular weight? What experimental condition causes multiple peaks in panel B to elute as a single peak in panel C Propose a reasonable scientific hypothesis about the biochemical basis for multiple peaks in panel B, but a single peak in panel C. Which peak in panel A contains protein(s) with the highest affinity for the Ni+2 Agarose column?

This is the principle of Protein Movement.

The movement of any proteins depend upon 2 factors, "the Charge of the protein " and "the lining of the protein ".

So,for analyzing any protein we use 2 types of gels with different Ph,the stalking gell and a resolving gell.

in this example the stalking gel is panal c with 5.0 Ph, and the resolving gel is panal b with 7.0 Ph. the different ph values help the protein to line up in the correct way and so they can be easily identified based on their molecular weight.

Procedure :-

before performing any sds page, we use Tris Hcl Buffer and the glycine buffer,so these two buffers are applied on both the plates,Now Hcl buffer releases chloride ions (cl-) and glycine can release zitter ions and acidic ions its movement differs according to the ph.

Now in panal c with single peak is because of glycine and protein movements are slow at the ph 5.0 where as at ph 7.0 the glycine and chloride are seperated so they form multiple bands differentitating proteins,chloride ions and glycine.

so a single band indicates the mixture is with both glycine and proteins and a multiple bands indicate protein,glycine,chloride ions.