After running E. coli transfection protein (molecular weight 15 kDa) on SDS-Page and performing Coomassie brilliant blue staining, using 15% separating gel and 3% concentrating gel,staining with 50ml staining solution A+B for 2 hours, and decolorizing overnight with MQ. I want to use different concentrations of BSA to draw a standard curve to get the standard equation, analyze it with Image J, and then calculate the E. coli transfection protein concentration.What voltage and current should I choose?
Dear Liang Tang
first of all, if you can buy an acetic acid and methanol free staining solution, i suggest to you to use the microwave owen to accelletare the process. You really do not need to do overnight destaing.
https://www.blogger.com/video.g?token=AD6v5dwhmY5GkO9_ezq3Va_Tfqi6u0ys99hBWy2OxXI0esS7px1EdB64LQXElfRjWFrifwHtPdDxUERBgln233r-2MAurJXhLDWcqkP1X5Gr1gSvjYZLKNS5fdeNossEHQrrnebrxiHw
regarding the voltage, it depends a little from the buffer that are you using (e.g Tris-glicine, MES, MOPS) and the strenght of your prower supply. However normally with standard SDS page costant voltage in the range 150-200V is ok. Lenght of run could be beetween 40 and 1h. I suggest you to use a pre-stained marker so you can monitor better the separation and decide when is it better to stop the gel.
best regards
Manuele
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