SDS Page too low voltage for a given constant current

[lakhoobhai]

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.

[butters]

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.

[skumarmadhu]

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

[lakhoobhai]

Originally Posted by butters

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