As you can see the bands are separated at the top but they are distorted at the bottom and there is more stain at the bottom while the positive control is at the top. when i satined the gel with glycosylation i got the same pattern, the positive control in pink at top and very light band of proteins at the top but highly stained and distorted bands at the bottom.. as i was getting light bands for glycosylated proteins at the top of the gel so i run the second time by doubling the protein concentration i got the same pattern but this time bands at the bottom of my positive control too… what could be the possible reasons to cause such pattern?
There is probably a large amount of lipids in your samples. They run at the bottom of the gel and produce bands that look like that. It may be necessary to clean up your samples by precipitation and washing of the pellets before dissolving them in SDS-PAGE sample buffer.
Hi,
It looks like your proteins are highly negatively charged in your samples. Due to glycosylation..? It could change migration ..
Probably also additional factors as Adam said ?
You did not mentioned which type of sample and proteins you wanted to separate and which type of gel. Probably SDS page..
1) What MW range are you interested in? You should be adjusting the gel density upward to get better separation on low MW.
2) Are you sure that you incubated the proteins long enough in the SDS/LDS sample buffer. There are some proteins that need more heat 95 C for 15 min to be fully coated with SDS/LDS, not the "recommended 75C that Invitrogen was advertising for LDS.
3) Are you sure that the gel was firmly attached to both plates across the full surfaces. If you have a gap along one plate, the proteins will run into the gap and just migrate to the bottom. If using old precast gels, sometimes a shock during shipping at just the right angle can make the gel separate from a wall. Dessication during storage can also cause this to happen.
4) Sample prep. Are you sure that you didn't precipitate or allow proteases to digest the proteins. Precipitation can sometimes be overcome with SDS and heat, but not always and usually manifests as protein left in the well with streaks in the lanes. Protease digestion can give you what you see and won't be reproducible between samples.
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