I am working on snake venom proteins and wanted to resolve it on SDS PAGE. I have attached image of my gel. I am getting a smear. Is there any way to resolve it better?
Sorry, but which lane do you think is smeary? I couldn't make that out from the given image. There are some sharp bands in your image. Depending on how the smear looks, it may be attributed to same protein getting posttranslationally modified (for e.g. multiple phosphorylation or ubiquitination). If you regularly get the same kind of smear for that particular protein, it might be the case. Or else, may be due to protease contamination, the protein has been degraded which may appear as smear on the gel. Also, in general, I think you can increase the current for electrophoresis so that you get sharper and crispier bands (reduces diffusion). You can go through the following link.
Hope it helps!
https://www.researchgate.net/post/Why_do_the_lanes_in_an_SDS_PAGE_tend_to_spread_outwards?_tpcectx=profile_questions
The smear in your ladder for lane 1 might be attributed to the voltage settings you've set. Run the electrophoresis for longer at a lower voltage - you will get better resolution. The smearing in lane 5 seems consistent with that
The smears in lanes 2-4 at the bottom of the gel are likely attributable to an overrepresentation of that protein in the sample (large concentration of a protein makes that fat band smear)
I wouldn't be too upset about this gel if I were you.
Try to load less protein, overload may be the cause. But in general, gel looks good.
Make buffers very precisely - check pH. Change tris on bis-tris (ph 6.8) in buffers for stacking gel and loading sample buffer.
I think your gel and your settings (voltage) are quite ok, because the marker and especially the heavy proteins there look very sharp. maybe another or fresh marker might help, most often modern marker from the common companies are not so good as compared to markers from more expensive companies (e.g. try Roche or calbiochem).
for the samples: looks to me not like post-transcriptional modification, this leads usually to a non sharp band in both direaction, up and down. as your bands smear only up, are therefore "hold back" to appear heavier, there is a contamination in the sample, or the stacking gel is not ok as @Mateusz mentioned.
1. simple try would be first to centrifuge the sampes for 30 min at max speed. this would remove lipid inpurities and heavier genomic DNA.
2. then try to increase APS and TEMED by 50%
3. and try to use a longer stacking gel.
4. cool the gel chamber with cool packs in the buffer reservoir.
5. if the problem persits even after centrifugaton, maybe more likely there is another contamination in your samples. lighter nucleic acids like RNA fragments, or an organic solvent, or something changing the pH in your laemmli buffer. try 2x buffer or increase the tris concentration.
sorry, it is a bit playing around. you could also use a protein purification method to remove all the stuff:
http://wolfson.huji.ac.il/purification/PDF/PAGE_SDS/NOVAGEN_Prepare_Sample_PAGE_SDS.pdf
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