transfer conditions in western blot - (Jun/04/2008 )

Hello. I'm having difficulty obtaining the expected results from western blot. I suspect that my transfer conditions are not good. The size of my target proteins are about 25 and 14 kD, and I'm using 140mA for 2 hours. Please give me some suggestions. Thank you.

-tenuissimus-

QUOTE (tenuissimus @ Jun 4 2008, 02:52 AM)

Hi,

I'm working with small proteins as well...can't tell you the mA because I always use constant Voltage. 10 V per minigel for 30 min. Otherwise I had the problem that my proteins were transfered through the membrane onto the filter paper...

Stardust

-stardust-

I use 0.8 mA per square cm of gel for 1 hour.

-K.B.-

We use 40 mA per gel, for one hour. Maybe your transfer time is too long and your protein pass through your membrane..

-Madrius-

Did you check the pore-size of your PVDF/NC membrane? Wondering if the pore-size is too big to have allowed your proteins to pass through?

-shyn-

Shyn, ~65 kDa protein has ~7 nm diameter when membrane has 0.22 um (220 nm) or 0.45 um (450 nm) pores.

-K.B.-

I am working with small protein too. I use 100v and 0.23AM for 45 min to 1hr. If I use more my protein will go out.

-saraarasus-

well how is your protein size marker ? do you get all the bands ?

2h seems too much for me. 30' to 45' of transfert is generally sufficent.
Moreover with little proteins, they tend to cross the membrane.

-fred_33-