wikiro Posted November 3, 2010 Share Posted November 3, 2010 In lab I was doing a His-Tag of FAAH SDS-PAGE and used commassie blue stain geling afterwards this then turned out to have a lot of bands that go all the way down to the front. I believe I messed up some where. I don't know if it has something to do with the gel, purification, or both. Can anyone help me on this? Ill attach the picture. We ran 2 gels and used them separately for the western blot (no number) and the commassie Blue (no name). Link to comment Share on other sites More sharing options...
CharonY Posted November 3, 2010 Share Posted November 3, 2010 I assume all lanes contain His-tag purifications? In the first pic the fifth lane appears to contain something else, whereas the rest could be just different dilutions. Also, a marker would be helpful. In the first gel the many bands may indicate incomplete purification (i.e. contamination with the proteome). Is the second pic a blot with FAA-specific antibodies? Link to comment Share on other sites More sharing options...
Horza2002 Posted November 3, 2010 Share Posted November 3, 2010 Im no expert on SDS pages only having done a couple....but I have had the problem that my protein degraded after purification and so I ended up with lots of bands in the individual lanes. Link to comment Share on other sites More sharing options...
CharonY Posted November 3, 2010 Share Posted November 3, 2010 Yes, that was why I was asking for a marker. If masses are found above the expected, chances are that it was not degradation. However, strongly degraded protein samples tend to give more smear-like images rather than the many bands visible. Link to comment Share on other sites More sharing options...
granovski Posted February 10, 2011 Share Posted February 10, 2011 As i see, your product isn't well purificated, your purity is around 1 to 2%. To determinate the purity, you must evaluate the active band on WB in comparison to the SDS-PAGE gel, anythig that has no signal are impuritys. A Marker is also needed, but the conclusion can be made without it. Best of luck Link to comment Share on other sites More sharing options...
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