sds page faint bands

[vishwanathiari]

I am unable to get good bands in sds page i use 5% stacking gel and 12 % speparting gel, sample was denatured for 10 min at 90 oC satined in comassin blue for 30 min and destained overnight i have attached phot of gel 1st and last lane are markers lane 1,2,3,5,6,7 are protein samples 20 micro liters was loaded in biorad mini conatining 4x sample buffer diluted there is continues blue background in the lane i dont know why its is happening i ran at 60 volts constant for 3hrs aprox
please help me thank you

[admin]

Looks like you have gel problems - specifically resolving during gel sepearation as your bands are not very sharp

I would make sure you run at constant current, and mix everything well for gel creation. Use fresh buffers, and make sure you use a stacking gel

Very important: How are you preparing your samples? Fresh Beta-mercaptoethanol? Also how are you preparing your loading buffer are you using Laemelli?

[protosome]

Indeed, how are you preparing your sample? I have had this problem when precipitating supernatant proteins with TCA and resuspending in laemmli. However, my cytosolic proteins from SDS lysed cells are perfect. I suspect is has something to do with the resuspension or solubility of the proteins. Or, it could be an interfering agent in your sample buffer.

[Elizabethsxw]

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