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Tricine gel problem


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#1 Zaax

Zaax

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Posted 31 January 2005 - 04:19 AM

Tricine coomasie stain


hi all

first question for me here.
please take a look at my image.

i made a standard tris-tricine gel in order to see expression of two small mol wight proteins that i hope i sucssesfully transfected into L8 cells.

this was my first tricine gel.

i then fixed it using gluteraldehyde and then stained with coomasie blue. yes i know coomasie is not the most sensitive method, i just feared i might have been losing my 7 and 9 KD proteins somehow in the transfere.

anyway it seems as if all the proteins are gettign stuck before reaching the 20 kd marker.

i might have added too much protein having loaded about 60 micrograms.. but the upper sizes seemed to resolve nicely. so i dont think thats the problem.

i used according to instructions, both a cothod and anode buffer etc...

i ran them for about 2.5 h @ 50-60 mAmp.

please any ideas? i wanted to see the expression of my protein domains.

by the end the buffer was slightly warm, but not hot...

ty

zaax




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