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Hello all, I have used stretched polyacrylamide gels successfully many times to collect RDC data for proteins. The last time, however, was several years ago. Now I am wanting to do this again, but I have encountered a problem I can't seem to overcome, one that I have never encountered in my previous gel work, namely that my HSQC spectra of the gels (no protein present) contain streaks in the proton dimension that I can't seem to get rid of. At first I thought I simply had unreacted acrylamide or other chemicals from casting the gel, but I have reproduced this problem many times now, even after soaking/washing the gels for 5-7 days after polymerization so that any unreacted chemicals can/should diffuse out. I'm providing the recipe I use for the gels, as well as the composition of the rehydration buffer, plus a screen shot of the HSQC spectrum. Anybody have any idea what could be the cause? I've tried different brands of acrylamide, different ratios of acrylamide:bis-acrylamide, etc., but these streaks are always present. Gel recipe (1 mL of 6% acrylamide gel): 0.79 mL H2O, 0.2 mL 30% polyacrylamide, 10 microL ammonium persulfate, 0.8 microL TEMED Gel rehydration / protein sample buffer: 10 mM MES (pH 6.0), 1 mM EDTA, 5 mM DTT
Any thoughts? Thanks. |
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What you are seeing here are signals of NH2 groups from polyacrylamide, not unreacted acrylamide or bis-acrylamide. This is described in one of the original RDCs-in-gel papers together with a modification to the IPAP-HSQC sequence to suppress NH2 signals (http://dx.doi.org/10.1023/A:1012417721455). Are you using the same pulse program as previously? And did you try to make a sample with protein to see how much the background NH2 signals would interfere with your protein's signals? |
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Thank you, Jiri, for pointing this out. I knew it of course had to be something relating to the acrylamide (it's the only chemical in the sample with amide groups!), but I kept thinking it had to be unreacted acrylamide somehow, because I never observed this before. Indeed I am using a different pulse sequence than the one I used before. My old university had Varian spectrometers and pulse sequences, but my current university has Bruker spectrometers and pulse programs. I will compare the Varian pulse program I used before with the Bruker one I have now to verify that the Varian sequence does include suppression of the polyacrylamide signals, while my current Bruker pulse program clearly does not, because I do see the streaks in my Bruker IPAP-HSQC spectra. The streaks are at a comparable intensity to my protein signals, but this is because my protein cannot be concentrated to any great extent. Thanks again. |
