Has anyone had issues with protein band smearing? I reduce the amount of sample I load on the SDS PAGE gel, but all that changes is the intensity of the smear. My protein standard runs normally. I incubate my protein on ice for 30 minutes in the presence of 100mM DTT, which has no affect on the smearing pattern. Any advice/suggestions would be greatly appreciated.
Dear Brittany, the problem is unclear due to the kind of smearing you have observed. You can use this link, just to identify better your problem and try a solution. Best regards,
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/smear.html
Membrane proteins are notorious for smearing up Western blots. In my experience, its not the membrane protein itself but all the other insoluble stuff in the sample, for example lipids. You could try TCA precipitating the sample prior to loading it on the gel. TCA precipitation has several drawbacks. Weakly expressed proteins can be lost, and it is not terribly efficient at precipitating low molecular weight proteins. Hopefully, these caveats won't be issues for you. Good luck.
I also agree with George Nieman. It is due to contaminations in proteins, may be lipids or some other insoluble stuffs. centrifuge at full speed (15000 rpm) for a while before loading and then carefull load top fraction. you can ultracentrifuge your protein solution at 100000 rpm and take spnt. The TCA precipitation may also help,if protein dont loss its activity.
Did you loaded other protein as a control to confirm that only your that membrane protein have the smearing problem? If you are sure about 100mM DTT that have no effect on protein then probably your buffer is problem if you are preparing gel by yourself. Check the pH of Tries(1.5M or 1M) for upper and lower gel. For lower gel use pH 8.8 and for upper use 6.8 exactly. Good luck.
When you've found a smear of proteins in the SDS Gel, it means that your proteins is degraded. Watch on your Lysis buffer and the conditions of lysis. My suggestions: 1) Use a complete composition of protease inhibitors in your lysis buffer. 2) Just load from the supernatant of the lysis solution.
Good Luck.
I think both the possibilities can be there either contamination or protein degradation. So better you check your buffers pH and use right combination of protease inhibitors. Also, carefully pipette out the supernatant without contamination of other undesired stuff. Good Luck...
Thank you all for your terrific suggestions and advice. I don't think my issue is a product of excess amounts of lipids. I don't always have the smearing problem. The problem started when we stopped using pre-cast gels and started making our own. My vote is to go back to pre-cast ;) (says the grad student not having to write grants). I just learned that you aren't suppose to pH the running buffer or transfer buffer, which I've been doing. I'm doing a Western today, so I will know tomorrow if my smearing problem was because of the buffer. It is also possible I'm having a degradation issue. I isolate the proteins on ice and use PMSF, so my hope is that isn't my issue. I wouldn't know how else to prevent degradation.
The concentration of salt also influence in the appearance of the band.
If your protein is diluted in a buffer who has a high concentration of salts, this could be the problem.
like M. G. Sharoar said, try to load a protein control, like a pre stained protein marker.
First of all check the purity of protein by running SDS-PAGE alone. You should get clear band of your membrane with designated MW. Then, immediately go for Western blot so chances of degradation of protein quality will be less. While transferring protein to membrane, gel and membrane should be intact and try for transfer at low voltage for quite long period.
My dear friend, as other friends suggested, it could be protein contamination, or could be fault in gel preparation. Other wise try to run it for little longer time with low voltage.
Dear Brittany, it looks that you are on a good way to resolve the problem. I have sometime "smears" in my loaded samples when I have too much RNA/DNA. I add DNAse and RNAse to my lysis buffer in my sample before I boil it for about 5 min, and it does the trick. Or the cheapest and laziest version: as soon as I get it out of the boiling, I place them in ice. I think that way I don't allow the DNA / or RNA to "refold" properly and is not that sticky. The other option: Could it be that your protein is glycosylated? I am not sure if you mention the production host for your protein. Glycosylation makes the protein bands look VERY wide (almost like a smear). Good luck
Dear Brittany, the problem is unclear due to the kind of smearing you have observed. You can use this link, just to identify better your problem and try a solution. Best regards,
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/smear.html
Brittany,
If you do not have the smear in precasting gel means that the problem is in glasses of your gel. Do you clean them with Aceton , EtOH or some other solvent? Precasting gels are in plastic
I suggest to try the gel running with low voltage for a long time. good luck.
If it is a highly glycosylated protein it can have a variable molecular mass and thus appear as a broad smeared band .
Try to use 5x even 10x of the normal quantity of DTT or beta-mecaptoethanol and incubate at 45 oc for 30 minutes before loading on the gel.
Dear Luisa F. Jimenez How we could solve the problem due o Glycosylation,
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