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Western blot transfer weak


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#1 GFAP

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Posted 24 September 2010 - 01:38 PM

Hi,

I think my western blot transfer doesn't work well. I Do the wet transfer overnight (invitrogen Xcell II module) with constant Volt as manual say. I see everything with my Red Ponceau but when I use ECL and see my films the central bands are weaker than the others. I use Tubulin but is the same. In twelve bans of tubulin of same lysis the central bands are weak.

Someone know why?

thanks

#2 protolder

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Posted 26 September 2010 - 09:51 PM

Hola, I´m not sure have your answer, or that I say you know. By the same way that little proteins run faster than bigs ones in a PAGE, the same occurs for transfer them from the gel to the membrane in WB. So if your protein is small you could short the transference time but if your protein has 100Kd or more, you have elongate the transfer time. I don´t knov the Invitrogene device´, but when I worked with te Bio-Rad wet system if you make a gross sandwich and have some difficulty to close it before put it in the cuve, the sandwich seems like (), so in the middle area the transference could be innefficient. At least this has been confirmated by my own experience and I hope serves to help you. Good luck




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