Run SDS page in various conditions: 1) As such 2) Add protease in outside large

Question

Run SDS page in various conditions:

1)      As such

2)      Add protease in outside large external domain (peripheral protein), then spin and run the gel on pallet.

Ques: What will be the banding pattern? (SDS PAGE)

In an Out/out vesicle, there is a large external domain on outside and trans-membrane domain inside.

single polypeptide

In an In/out vesicle, there is a small external domain on outside and large trans-membrane domain inside.

1) What will be the banding pattern on SDS PAGE if protease is added on outside of i/o vesicle and the spinned and run the gel on pallet.

2) Salt wash and the spin and run gel on pallet

Explanation / Answer

1. When we subject a cell homogenate to SDS-PAGE, then the proteins are separated based on size. 2. when we treat peripheral protein with protease, the peripheral protein is lysed. There will be no bands as the protein has been lysed. ----------------------------- 1.If protease is added on outside of the in/out vesicle and then run in a gel, the outer domain is lysed while the inner domain is intact. Hence the gel shows the presence of the inner domain only[only one band]. 2. When we wash with salt and then run the gel, the high concentration of salt in the sample creates artifacts in the gel. Hence you may see extra bands.

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