You hypothesize that your protein of interest moves from the cytoplasm to the nu

Question

You hypothesize that your protein of interest moves from the cytoplasm to the nucleus when a cell is heated from 37Celcius to 42Celcius

1) Design a microscopy experiment to test this hypothesis. Please include the labels and type(s) of microscopy you would use. Also, please draw illustrations of what you predict your experiment to look like.

2.) Design an experiment to test this hypothesis using cell fractionation and SDS-PAGE. Please include what samples you would collect to run on the gel, and how you will identify your protein of interest. Also, draw an illustrationof what you predict your gel to look like.

Explanation / Answer

1.

To observe the protein of interest (POI) into nucleus, we use following steps:

Protein is present in cytoplasm; stain it before heating by using fluorescently labeled antibodies.

Observe the presence of protein and note it.

Now stain cells by Nup49 or any nuclear importing protein dye and heat the cells. Observe under fluorescent microscope. We can see clear results whether the protein of interest has moved or not.

2.

To observe the protein of interest by using SDS-PAGE, we use following steps:

Firstly, isolate total protein from cell.

Obtain protein by ligand mediated affinated chromatography.

Now tag the POI with avidine-Hsitidine (avi-His) or any other tag protein.

Now initiate SDS PAGE process. We have to run three columns. One is for marker, second is for control and third for POI-avi-His. For control, we would take Bovine serum albumin. Stain, control with normal GEL dye and POI-avi-His with streptavidin.

Run proteins on Gel and observe under UV light.

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