I have done a myoglobin extraction using salting in/out, dialysis, and size exclusion columns. When running the extraction on SDS PAGE against a low MW ladder I get the following results (pic attached). Myoglobin is around 17 kDa and the band to the far left is supposed to be ~15 kDa (2nd blue band) The green band is ~10kDa. Instead of being above the marker the band is below and split into three closely positioned bands. What could be going on? Bad ladder?
Previously I had done the same extraction and the band was on the same level as the ~15 kDa marker and confirmed via mass spec that it was myoglobin. Nothing is making sense.
Maybe you sample has been denatured. Have you analyzed these bands again via mass spec?
Some proteins have this behaviour... They do form multimers even with SDS in the gel, which leads to a "ladder" pattern. Moreover, if the protein - for some reason - does not bind SDS homogenously, you get the'll the wrong size. My advice would be to change the gel porosity (you can test 10, 12 and 14 %) to test wether the size changes with porosity (which can sometimes happen too).
I agree with Fréderic. You may also try and obtain your extracts under denaturing conditions (i.e. with SDS or TCA) and check that your sample is not being proteolytically degraded. Do your MW markers give a neat straight line on a semilog plot of MW vs. Rf?
Dear John
I agree with both Dr. Khalid Mohammed Naji and Dr. Frédéric Fischer. you'd better try the different prosity of gel including 10, 12 and 14%. Furthermore, check the pH of your buffers precisely before carring out the SDS-PAGE.
Dear John
I agree with both Khalid Mohammed Naji AMD and Florencio E. Podestá.
Because your target protein has a low molecular weight, using high percent prosity of gel could be more effective and protein marker (Pageruler) contains purified proteins with different weight that maybe their movements on gel slower than the extracted protein. Therefore, Extracted protein may not move exactly with the purified protein.
Did you protect your proteins from proteinases? Is pH of the buffer you used appropriate? Did you have a controls on the same gel (I mean some close to mass a different protein with known characteristics?).
- Badges
- Science method