Searching for Proteins: Staining Cells

Another method for looking at protein expression is to stain cells for the specific protein. The following procedure goes over how cells are stained.

1. Cells are fixated, so all the proteins become linked so that nothing moves.

2. Cell are treated with 0.5% triton for ten to fifteen minutes

3. Wash or rinse cells with PBS three times and add 3% BSA stock (blocker)

4. Place the cells on a rocking plate and wait for about an hour

5. Rinse and add primary antibody, leave over night.

6. Rinse and then add secondary antibody

7. Image!

So first the cells are fixated so then none of the proteins can move around, they are all covalently bonded to each other in a complex pattern. Then, the triton permeabilizes the cell, which makes it more likely to let statins and dyes go into it. Jon was planning on staining caveolin by using antibodies that specifically bind to it. But first, the cell must be blocked with BSA solution. BSA weakly binds to all the proteins inside the cell. This way when the primary antibody is added, it doesn't weakly bind to all proteins, instead, it strongly binds to one protein (caveolin). After the primary is added, then a more general secondary antibody is added to the plate. The secondary antibody is more general in that it binds to a specific animal’s antibody. The secondary antibody is fluorescent. The amount of light emitted from the sample is supposed to represent the amount of caveolin.

This method is neat because it allows the person to visually see protein expression in cells, but small sample sizes and bias can unintentionally play a big role. But at the same time, it is less complex than a Western Blot and allows the scientist to view protein expression while the cell is still mostly intact.