Hi Bhaskar,

Did you try not heating the protein before running gel? You can give it a try once. I guess you are working with a membrane protein and some membrane proteins have this behavior. Also, where do you expect the protein band? if it small then this could be a case of lipid/detergent based association. Do you know if your protein has any kind of PTM which can also give such patterns? Sorry for asking so many questions but in an unknown system you need to address such things. Good luck with your experiments!

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Best,

Kalpana

Hi Kalpana,

I am working on cytosolic protein. In soluble fraction, I able to get smooth bands without streaks. Recently, we try to see our protein in exosomal vesicles, then I start getting this problem. I am using sucrose to preserve the vesicles. and to prepare sample for western blot, I simply boil them in Laemmli buffer.

Hi Bhaskar,

Do you know if sucrose also binds to your protein? For this, you can try not adding sucrose or using a lesser amount if it is critical for vesicle stability. Did you try not using heating your samples?  Also, your situation suggests that something very specific is happening so it can not be any contamination. You can once try loading the samples with vesicles without heating as well to check if the phenomenon is related to lipid effect on your protein. It is also a possibility that your protein starts showing anomalous behaviour on SDS PAGE. I will update you if I get to know more about any such system. 

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Best,

Kalpana

Hi Kalpana,

Thanks for your suggestion. I try and update you for the same

Thanks

Could be due to glycosylation or sialidation of that particular cluster of proteins. Try treating with glycosidases and/or neuraminidase prior to running your gel.

Hi Skinner,

I do not about glycosylation or sialidation, As predicting tools rule out these possibility.

Thanks

Bhaskar