Ionic line in 2D gel - troubleshoot (Apr/17/2007 )
Hi all,
Need help with my 2D gel. I've been getting the ionic line on my 2D gel. The problem is this line is only appear on top of the bromophenol line after i stained with staining solution which is very hard for me to determine when to stop my 2D electrophoresis. The distance between the bromophenol line and the ionic line varies from time to time. Sometimes, it even appear in the middle of the gel which in this case all the spots below the line will not no be appear. So, could anybody please help me to troubleshoot this problem. Is it because of my agarose sealing gel. Thank you.
Regards,
cattoyee
Need help with my 2D gel. I've been getting the ionic line on my 2D gel. The problem is this line is only appear on top of the bromophenol line after i stained with staining solution which is very hard for me to determine when to stop my 2D electrophoresis. The distance between the bromophenol line and the ionic line varies from time to time. Sometimes, it even appear in the middle of the gel which in this case all the spots below the line will not no be appear. So, could anybody please help me to troubleshoot this problem. Is it because of my agarose sealing gel. Thank you.
Regards,
cattoyee
do result the differences in 2D gel performance from different ionic strength of your samples? or other reasons?
are you soaking your first dimension gel in sample buffer long enough?
how old and used is your sample soak buffer?
try fresh.
how old and used is your sample soak buffer?
try fresh.
Hi,
Do you mean rehydration time? I did my rehydration for 16 hours and usually the rehydration buffer was kept in -20C not more than 2 weeks. Is it because my rehydration buffer or is it possible because of my agarose sealing gel was spoiled?
thank you
Regards,
cattoyee
Do you mean rehydration time? I did my rehydration for 16 hours and usually the rehydration buffer was kept in -20C not more than 2 weeks. Is it because my rehydration buffer or is it possible because of my agarose sealing gel was spoiled?
thank you
Regards,
cattoyee
actually, i mean sample buffer soak time between dimensions. this removes ampholytes (if you are running a mobile pH gradient) and reacts your proteins with sds so that they are ready for the second dimension. if not soaked long enough then you will have an additional buffer in your system (from your first dimension gel) and that can cause a line in your gel.
actually, i mean sample buffer soak time between dimensions. this removes ampholytes (if you are running a mobile pH gradient) and reacts your proteins with sds so that they are ready for the second dimension. if not soaked long enough then you will have an additional buffer in your system (from your first dimension gel) and that can cause a line in your gel.
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Hi mdfenko,
Thank you for your informattion. I will try to do it and see if I can remove the line. Thanks again
best wishes,
cattoyee