Weak western signal for help - (Jul/23/2010 )
mdfenko on Tue Jul 27 13:58:50 2010 said:
transfer with caps is fine. you may want to determine if you are transferring too long, at the higher pH the proteins may be moving faster in the electric field.
what is the pore size of the pvdf and the size of the proteins of interest?
if your proteins are small then you may want to use a pore size of 0.22 um or smaller. if you are using 0.45 um then you may want to try a backup membrane to ensure that you are not blowing through (also, stain the gel to see if any is left behind).
Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with commassie blue from Biorad, there are definitely bands on the gel after transfer.
The western is really puzzled, I tried same samples when I use NuPage from Invitrogen, the signal is more than 10 times different.
-mickey-
mickey on Fri Jul 30 01:13:34 2010 said:
Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with coomassie blue from Biorad, there are definitely bands on the gel after transfer.
so you are not getting efficient transfer? are all the bands still in the gel (including the 9kDa)? (i would think that the 9 kDa protein would pass out of the gel rapidly and may pass through the membrane)
did you equilibrate the gel with transfer buffer before assembling the transfer sandwich (not always necessary but if you are not successful without it then you may want to try or vice-versa)?
did you ensure that there is full contact between the gel and membrane?
-mdfenko-
mdfenko on Fri Jul 30 20:24:23 2010 said:
mickey on Fri Jul 30 01:13:34 2010 said:
Thank you so much. I use 0.22um PVDF or nitrocellulose and my protein size ranged from 9kDa to 55kDa. The problem is when I stain the gel with coomassie blue from Biorad, there are definitely bands on the gel after transfer.
so you are not getting efficient transfer? are all the bands still in the gel (including the 9kDa)? (i would think that the 9 kDa protein would pass out of the gel rapidly and may pass through the membrane)
did you equilibrate the gel with transfer buffer before assembling the transfer sandwich (not always necessary but if you are not successful without it then you may want to try or vice-versa)?
did you ensure that there is full contact between the gel and membrane?
Thank you. Only bigger than 30kDa stained on the gel. I alwasys equilibrate the gel for 15 to 30 minutes. Also I cutted a 10ml pipet to get rid all the bubbles.
Now I could get good signal from 10% and 12% gel, also bands I want from 15% gel with little background. Problems are I now do some overexpression the signal seems not so good on 15% gel. Although other people's data are from NuPage or Tricine system, just because I want 9kDa and 70kDa on the same PAGE.
-mickey-
mickey on Sat Jul 31 01:37:32 2010 said:
Thank you. Only bigger than 30kDa stained on the gel. I always equilibrate the gel for 15 to 30 minutes. Also I cut a 10ml pipet to get rid all the bubbles.
Now I could get good signal from 10% and 12% gel, also bands I want from 15% gel with little background. Problems are I now do some overexpression the signal seems not so good on 15% gel. Although other people's data are from NuPage or Tricine system, just because I want 9kDa and 70kDa on the same PAGE.
you may want to try a gradient gel to visualize 9 and 70 kDa on the same blot.
-mdfenko-