Each of these washes will remove some of the more stubborn proteins that don't interest you. The most popular His-tag carries 6 histidine and proteins carrying these tags generally elute when the imidazole concentration reaches 100 to 180 mM, which means you can do washes at 30 mM, 50 mM, 70 mM and 90 mM imidazole before eluting your protein with 500 mM imidazole buffer. Hope this helps!įinally, the most obvious thing to do to clean up your prep is to do more washes with higher imidazole concentrations before you elute your protein. Do a test - check the pH in the upper and lower buffer chambers after a run. I would try filling the lower chamber completely and/or increasing the buffering capacity, as well as limiting the amperage a little more. It could be simply too much current causing this (which would increase the production of H+ at the anode), or that your buffer needs more buffer salt.
This buildup of positively charged hydrogen ions will begin running up the gel towards the negative electrode causing what you see. Remember the anode reaction for electrolysis: 2(H2O) -> (O2) + 4(H+) + 4(e-). My hunch is that after a long run there is a buildup of H+ in the lower buffer chamber that exceeds the buffering capacity at some point, reducing the pH. Not very scientific, but the problem never came back (and I have better things to do than run experiments on why). I tried remaking gels, but it was always the same - until I started making my 1X MES with a slightly heavier hand so that it was a little more than 1X. My assumption is that it has to do with the pH in the lower buffer chamber. It usually happened lower on the gel near the end of the run. 12% Bis-Tris gels running MES (also homemade from 20X stock). For me this happened with batches of gels that I was casting myself. Unfortunately, I don't have any solutions, just some suggestions. Sorry for old fashioned way but I hope it helps.įinally somebody else has had this problem! I tried explaining this to people and usually just get silence. It may not give me the precise last digit of my protein pI but certainly give a hint on what pI it is about.
Further, I plot the activity (y-axis) to the pH values (x-axis) of samples recovered from each resin. At least I knew by then the relation between which band represent my enzyme activity. I checked on SDS PAGE and the protein bands are being corresponded with the activity assay. I took the supernatant and check for protein and activity (since it was an enzyme). Just incubate for about 1-2 hours (did mixing by inverting the tube every 10 minutes or so), and then spin down with simple bench mini centrifuge. I did the same with cation exchanger (CM). Then, I added 100 mikroliter of the protein solution into several tubes that each containing 250 mikroliter anion exchanger resin (DEAE) equilibrated with about 50-100 mM buffer at pH values of 4.5, 5.0, 5.5. I prepared the protein in a buffer of low concentration (10-20 mM), the pH is not that important. Something I have done over decades ago for my thesis: make use of ion exchanger resins.