Question

Outline the procedure (Steps) of Immunolabeling that you would use to identify the location of monoclonal protein within cells.

Answer 1

Ans:- Immunolabelling means labelled antibody are used to identify the location or interaction desired protein.

Steps involved in identification of location of monoclonal protein in the cells.

Step 1:- Take out the grown cells approximately 10 million cells. Centrifuge the cells.

Step 2:- Wash the cells with PBS to remove the left over media that may produce background fluorescence.

Step 3:- Fix the cells using paraformaldehyde for 15 minutes. Fixation of cells are required so that the physical state of the cells are preserved.

Step 4:- Wash the cells to remove paraformaldehyde.

Step 5:- Permeablize the cells with mild detergent for 15 min. Permeablization is needed so that your antibody should go inside the cells and detect protein. If you are detecting cell surface protein no permeablization is required.

Step 6:- Wash the cells. Incubate the cells with primary antibody that are against the monoclonal antibodies for 3-4 hrs.

Step 7:- Wash the cells to remove unbound antibody.

Step 8:- Incubate the cells with secondary antibody that are against primary antibody, and labelled with fluorescent tag.

Step 9:- wash the cells after 1 hrs.

Step 10. stain the nucleus with DAPI.

Step 11. Spread a few cells onto the glass slide.

And proceed to confocal or fluroscence microscopy.

There will see the location of your monoclonal protein due to fluorescence. In this way you can detect the localization of monoclonal protein.

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