PROTEIN BIOCHEMISTRY DAY FOUR (PART TWO)

While your gel is running, you will quantitate the amount of total protein in each of your purification samples using the Bradford (BioRad) assay. When your gel is finished running, you will "stain" the gel with Coomassie blue dye, and destain the gel to remove unbound dye. Your grad TA will then dry your gel for you and return it to you on Day 5.

BRADFORD (BIORAD) ASSAY

The first step in the Bradford (BioRad) assay is to assay samples of known protein concentration. You will use this data to create a standard curve.

Here is another view of a set of BSA protein standards assayed using the Bio-Rad assay protocol. Which sample contains the most protein? How can you tell?

Safety reminder: Because the Bio-Rad reagent contains phosphoric acid, it must be disposed of in the labeled waste container in the fume hood.


SDS GEL STAINING/DESTAINING/DRYING

Your gel is finished running when the tracking dye reaches ~0.5 cm from the bottom of the gel, as seen in this photo.

To remove the gel from between the two plates, first cut the plastic "wrap" on either side of the gel with a razor blade.

Then, slide the razor blade between the two plates, and pry the plates apart. Usually the gel will stick to one of the two plates.

Staining of gels should be carried out in the white plastic freezer box found in your bench.

Rocking the freezer box on the tilt table will ensure that the gel gets evenly stained.

"Destaining" of the gel will remove any Coomassie blue dye that is not bound to protein.

Your grad TAs will dry your gel after it has been stained and destained. This involves placing the gel between two pieces of cellophane soaked in a 5% glycerol solution (left photo), clamping the gel frame together (right photo), and placing the gels in the hood to dry overnight. Your dried gel will be returned to you on Day 5.

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