Examining Net Charge of Protein

(Like said in the About the Site, I’m not giving any further technical details and I don’t take any responsibility if someone tries to repeat this experiment. May this be a source of inspiration instead)

Without going into details, at work I wanted to find out the net charge of one protein. I didn’t care to find out how the other researchers in our institute might do this kind of experiment. That’s because it would still mean that I need to get familiar with new, unknown things, and because I like to get know-how skills right from the beginning. Furthermore, the coming setup is needed in other experiments as well. So, I built a small, symmetrical electrophoresis chamber from a 50 ml tube. I made an opening on one side and then glued the removed piece under the tube for giving support. I made dilute agarose gel in buffer with the pH where I want to know the charge of my protein. Then I casted and cooled the gel inside the chamber. I used a small plastic piece for making a sample well in the middle. After the cooling, I removed the piece, cut the ends of the gel and used suction for removing the heads. I dissolved my protein in the same buffer with glycerol and bromophenol blue, so that the whole mixture would settle easily in the bottom of the well and I would see where the liquid goes. I secured the whole thing in ice bath and put some aluminum foil, or electrodes, in the end cavities filled with the buffer. 100 V connected over the opposite aluminium pieces (giving about 60 mA) and after 7 minutes the bromophenol blue was migrated about 1 cm. I removed the cap from the tube, took the gel out, cut sample pieces from it and used a common staining method for revealing the location of the protein. An intense dark band formed only in the other side of the well, which highly suggests that my protein has negative net charge in the used pH. I still need to do this without the bromophenol blue, but as far as I understand it should not pull proteins with it. Here are some photos from my experimental setup.

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