Uneven banding - Western blot troubleshooting - (May/29/2013 )
I'm new to SDS PAGE and am facing trouble with uneven bands. I've attached a picture pointing out some of these problems:
1) The band is raised upwards in the middle (like an up arrow).
2) Uneven bands, with more fluorescence at the ends of the band and less in the middle.
3) A white spot in the middle of a pack of bands.
4) Tapering of the band at the edge of the gel.
5) Half of the band is raised and more concentrated than the other.
This was 12% gel ran at 70V through the stacking gel and 190V through the separating gel. Transfer was done at 100V for 90 minutes.
I suspect many of these problems are due to the uneven distribution of ECL during the exposure step.
Any and all help is greatly appreciated! Thanks in advance.
1 and 5 - you tore the well when you took the comb out and this left the well uneven, which translated into uneven banding. Too high a salt concentration can also cause phenomena a bit like this, but that usually presents as multiple wiggles in the band. This can also be caused by distortion of the gel when transferring.
3 and 4 - I don't see a white spot - I see an overexposed image which means that your bands are not sharp! The curvature and tapering is due to you having run the gels too hot (i.e.too high a voltage/amperage).
2 is due to sample loading -practice practice practice...
Thanks for the explanations bob1.
A few questions:
- what voltage would you suggest to run the gel at?
- what exactly about sample loading was the problem? Is there a particular way that I should do it? (currently, I insert a 50uL syringe half way down the well and insert the sample)
I would run it at a maximum of 150 V.
Nothing in particular that I can identify without watching you load - however, make sure that you are using an adequate volume for the well size, and spin the precipitates out of your samples before loading (and make sure you don't load them).
also, make sure your wells are clean of any gel "skins" or blobs or particulates and the bottoms of the wells are flat, the teeth are evenly spaced and straight.
you can bring your loading needle to the bottom of the well (careful not to puncture the bottom) so that you don't mix the sample with the electrode buffer (layer the sample under the buffer). then remove the needle carefully so you don't disrupt the layer.
make sure there are no bubbles in the well or stacking gel.
make sure there are no separations between the stacking and running gels (can occur when you remove the comb).
make sure your apparatus is side to side level.
put blank samples in unused wells.
try not to use end wells (except for blank samples), if possible.