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posted Nov 20 '13 at 14:26

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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

RDC Gel Problem Image

Any thoughts? Thanks.

click to hide/show revision 2
No.1 Revision

posted Nov 20 '13 at 14:27

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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

RDC Gel Problem ImageRDC Gel Problem Image

Any thoughts? Thanks.

click to hide/show revision 3
No.2 Revision

posted Nov 20 '13 at 14:29

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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

RDC Gel Problem Imageimage : RDC Gel Problem Image

Any thoughts? Thanks.

click to hide/show revision 4
No.3 Revision

posted Nov 20 '13 at 14:29

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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:RDC Gel Problem Image

Any thoughts? Thanks.

click to hide/show revision 5
No.4 Revision

posted Nov 20 '13 at 14:32

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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:RDC Gel Problem ImageRDC Gel Image

Any thoughts? Thanks.

click to hide/show revision 6
No.5 Revision

posted Nov 20 '13 at 14:37

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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

RDC Gel ImageRDC Gel HSQC Spectrum Problem

Any thoughts? Thanks.

click to hide/show revision 7
No.6 Revision

posted Nov 20 '13 at 14:38

ChemMJW's gravatar image

ChemMJW
99

Streaks in RDC Gel Spectrum

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

RDC Gel HSQC Spectrum Problem

Any thoughts? Thanks.

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