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 protein 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.

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 protein 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.

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 protein 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

image :

Any thoughts? Thanks.

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 protein 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

image : image:

Any thoughts? Thanks.

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 protein 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

image:

Any thoughts? Thanks.

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 protein 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.

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 protein 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.