Hi experts,

I need very pure protein for FACE analysis. As last stage I'm going to use purification from SDS-PAGE: cut out single band from the gel and then extract protein. I'm afraid that SDS (still bound to my protein of interest after extraction) will interfere with PNGase F digestion (though reaction mixture contains SDS at final concentration 0.05%). Could you, please, suggest method to remove SDS from my protein sample after extraction SDS-protein complex from gel ??? By now I have found only 1. info on GeBa kit, but it needs lots of buffer changes, 2. guanidine-HCl purification, that needs several precipitation steps and in both cases I could end up with unsufficient amount of protein.

Thank you,
Larisa

Larissa,

Im not sure you need to "remove' the SDS at all. NP-40 detergent added to the PNGase digestion buffer should out compete the SDS allowing the reaction to occur normally. The mechanism is apparently unknown? Some information is available on the NEB PNGase F page.

http://www.neb.com/nebecomm/products/productP0704.asp

Rb

Dr. Bishop, Thank you very much for your reply. I want to provide you with more details. We need to send PURE protein to OTHER facilities (different city) for FACE analysis. The minimum amount they will need is 50mcg. Below are my calculations - correct me if I'm wrong. Let's say I'll have this amount of protein/gel band. Amount of SDS bound to protein will be 1.4 fold (ratio of binding 1:1.4), i.e. 70mcg. Digestion reaction (prior to PNGaseF digestion) will be performed 1st (other facility): in 50mM Tris-HCl 7.3, with final concentration SDS as 0.05% and beta-MEt as 50mM, total reaction volume 38mcL. If I have already SDS in my sample from gel, then without adding extra SDS and performing additional digestion, SDS concentration (in 38mcL without adding extra SDS) will be 0.18% (~3.7 times more than should be in digestion reaction). After digestion they add NP40 (final concentr 0.075%) followed by PNGaseF. That's why is my concern - should I perform 2nd digestion with extra SDS? Should I recommend to add more NP40 to inhibit 3.7 fold amount of SDS ? What will be the best way to handle the situation and have successful PNGasaF digestion??? Thank you very much, Larisa [quote=R Bishop]Larissa, Im not sure you need to "remove' the SDS at all. NP-40 detergent added to the PNGase digestion buffer should out compete the SDS allowing the reaction to occur normally. The mechanism is apparently unknown? Some information is available on the NEB PNGase F page. http://www.neb.com/nebecomm/products/productP0704.asp Rb[/quote]

Larissa,

Sorry for the delayed response. I was out of the lab yesterday for my bday.

I see your concern in this situation. I think your calculations are correct and you are in a "trial and error" phase of the project. Why not take a small amount of your protein and do a pilot digestion with PNGase F in the NP-40 concentrations you figured out after gel elution? Its really the only way you are going to figure this out. Keep in mind there are only three things you can vary in an experiment; time, temperature and concentration. My bet is by doing a reasonable pilot you can quickly figure this experiment out.

As an alternative, can you digest your protein with Np-40 prior to running the gel and cutting the band out?

Best of luck

Rb