To begin this procedure, Kumasi Brilliant Blue or CBG two 50 is dissolved in one liter of bi distilled water by stirring for two to four hours. Hydrochloric acid is later added to the dark blue solution with stirring for another minute, and the solution is stored in the dark for later use. Following protein sample preparation and STS page, the gel cassette is disassembled and the gel placed in a box for three subsequent washing steps with bi distilled water.
After the third washing step, the water is replaced with enough CBB staining solution to cover the gel in the box. The box is heated in the microwave for 10 seconds, and the box with the gel is then placed on a shaker. For completion of staining, protein bands can be observed after one minute.
Hi, I'm Amri Lawrence from the protein expression purification cough facility at Emberin Heidelberg. Today we'll show you a protocol for staining proteins with cooma brilliant blue G hundred 50 in chloride gels. Unlike classical staining methods, our protocol does not use any organic solvents or acetic acid.
We use this protocol to analyze our proteins after every purification step. So let's get started. To prepare the CBB staining solution, 60 to 80 milligrams of CBB is dissolved in one liter of bist distilled water by stirring for two to four hours.
After the CBV is dissolved, transfer the solution into a fume hood. In the fume hood, carefully add three milliliters of concentrated hydrochloric acid to the dark blue solution. With stirring for one minute gloves should be worn since the final solution will have a pH of two.
Once the solution is made, it is stored in the dark for later use. The solution can be stored for weeks or up to several months without losing its staining efficiency. To prepare protein samples for SDS page, appropriate aliquots of protein samples are mixed with loading buffer to a final concentration of one x loading buffer heat the protein samples for about five minutes before loading.
While the samples are being heated, the gel electrophoresis chamber is prepared for the run. We use precast gels in a mini cell with MES buffer as the running buffer. However, any other gel and electrophoresis system can be used.
The heated protein samples are then loaded on the gel and electro East for 50 minutes at 220 volts. When the SDS page is complete, the gel cassette is disassembled and the gel is placed in a box with 100 milliliters of bist distilled water. For the subsequent washing steps, the box is heated in the microwave oven for 30 seconds.
Heating should be stopped before boiling occurs. After microwaving the box with the gel placed on a shaker for three to five minutes, repeat this washing step twice with fresh water. After the three washes with water, CBB standing solution is added to cover the gel in the box and the box is heated in the microwave for 10 seconds without boiling.
After that, the box with the gel is placed on a shaker for completion of the staining. Protein bands can be observed after one minute and after 15 to 30 minutes. The staining is usually strong enough.
The staining solution is poured off and 50 to 100 milliliters of bi distilled water is added. In order to further detain the light blue background of the gel on a shaker, the water can be replaced by fresh water for further des staining. If needed, the gel can now be scanned, photographed, or dried for long-term storage.
A gel that has been stained following this procedure should look like this. In this gel, the protein bands are stained well and clearly visible. While the background is negligible.
The first lane indicated by the asterisk contains molecular weight markers. In contrast, a gel that was not washed sufficiently prior to staining will look like this In this gel, the washing steps were too short, so the protein bands were not stained well enough. Note that the lane indicated by the asterisk contains the same protein amounts of molecular weight markers as a previous gel, but shows less efficient staining.
We've just shown you how to stain proteins with co massive brilliant blue in polyamide gels without having to use any toxic or flammable organic solvents. When carrying out this procedure, it's important to remember that the washing steps are critical for the efficient staining of the proteins. So that's it.
Thanks for watching and good luck with your experiments.
