Hi. I am trying to study a biotin modification of my target protein in a crude cell extract and am interested to determine how much of my protein in getting biotinylated. After biotinylating my protein, I added streptavidin and ran a native PAGE. I see that the ladder is running fine, but most of the protein sample is stuck at the top of the gel. I transferred the gel to a nitrocellulose membrane, and probed it with my protein of interest. While I can see a mass shift, the bands don't look sharp, and also the size does not correspond to the size marker. Any suggestions?
Proteins do not necessarily run according to their expected size in a native gel. Rather, running behaviour depends on the charge-to-mass ratio. And, if using standard buffers, the protein will have to have a negative charge to migrate in the gel at all (it will go in the opposite direction if it has a positive charge). If you are close to the pI of the protein, it won't have a net charge and only move weakly by diffusion.
A second reason that the protein is not running but getting stuck in the stacking gel might be because of aggregation. In fact, this is the likely cause of the non-migration. Streptavidin has four binding sites for biotin, so you are creating large complexes that might not be able to enter the gel.
You could try running a Blue native PAGE which increases the negative charge on the protein but does not dissociate protein complexes. However, if your protein is being biotinylated on several sites, you could still be forming a network of proteins that still cannot enter the gel.
Thank you Jack for your suggestions.
Do you think I can run a standard SDS-PAGE (with BME) and still comment on the modified mass of my protein if I do not heat my samples (see attached photo)? The only problem is that the modified mass does not add up. My protein of interest is a dimer, where each monomer is ~25 kDa; Streptavidin is a tetramer of ~53 KDa (each monomer ~14 KDa).
Hi Faisal,
It looks to me like you can differentiate the native form of the protein from the biotinylated form using BME (and streptavidin?), as you get a nicely resolved band for the modified protein when you add BME. If your only objective is to determine the level of biotinylation, this does not appear to be very high as most of your protein still migrates as a monomer. How much streptavidin do you add? And equimolar amount?
I don't think SDS-PAGE will be able to resolve the modified and non-modified versions as the biotin is less than 1 kDa in size. I'm not sure whether the biotin-streptavidin interaction is stable in SDS in the absence of heating (there must be literature on this). If it is, you might be able to get a clearer picture of what's going on.
Mass spectrometry might be the way to go here.
I'm wondering what the application is that you would need to know the exact level of biotinylation?
Hi Jack.
I added 20ug of streptavidin for 125ug of protein lysate; one possibility for not seeing a more robust signal from the modified protein is that perhaps I am not saturating the mixture with enough streptavidin.
This study is aimed to identifying drug targets. I used a biotinylated form of the drug to isolate and identify proteins that it binds to using mass spec., and have seen a 40% reduction in the enzymatic activity of my target protein. The experiment mentioned here is to see if the reduction in activity corresponds to the amount of protein modification by the drug.
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