SDS-PAGE Help.

[wikiro]

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).

 

 

[CharonY]

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?

[Horza2002]

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.

[CharonY]

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.

[granovski]

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