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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


SDS Page too low voltage for a given constant current

SDS Page too low voltage for a given constant current - SDS-PAGE Gel Electrophoresis Forum

SDS Page too low voltage for a given constant current - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 10-12-2011, 01:40 PM
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Default SDS Page too low voltage for a given constant current



Hi I am doing SDS page analysis in fact western of b-actin from Hepg2 cell lines. I am facing two problems.

1. When I do SDS page the loaded sample moves very slowly. I am using BioRad Miniprotean. I use 30 mA for two gels but the sample doesent move at all !

The value of volt is very low. If I increase the value for the volt to 50 the value of the current increases to 300 mA or more ! I am using Tris-Glycine SDS buffer? It takes a lot of time and it heats up.

2. After transfer primary Ab and secondary Ab a lot of unspecific binding?! In fact all the proteins were stained.
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Old 10-13-2011, 04:54 AM
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Default Re: SDS Page too low voltage for a given constant current

I think there are something wrong with your buffer or mp3/tetra cell system. If you check the manual they recommend 200v and some lab prefer lower amp such 15-25mA per gel.

Tell us your gel preparation formula and your running buffer. and how your load. Thanks.
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Old 10-15-2011, 08:02 PM
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Default Re: SDS Page too low voltage for a given constant current

Run a gel at constant voltage (75), use 10%SDS gel for b-actin

do secondary antibody (1:5000) in 5% milk n incubate for 1 hr 15min, wash trice in TBST each 10 min
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Old 11-10-2011, 10:17 AM
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Default Re: SDS Page too low voltage for a given constant current

Quote:
Originally Posted by butters View Post
I think there are something wrong with your buffer or mp3/tetra cell system. If you check the manual they recommend 200v and some lab prefer lower amp such 15-25mA per gel.

Tell us your gel preparation formula and your running buffer. and how your load. Thanks.
Thankyou very much the problem is resolved
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