6 replies to this topic

[polaris]

I'm trying to use tris/glycine/20%MeOH (Towbin's) with 0.1% SDS to wet transfer onto PVDF. I think I'm blowing right through the membrane; my markers are not on the membrane or the gel after 100V for 2hrs.

If you use Towbin's buffer and a wet transfer system to PVDF (mine is bioRad mini):

What is your:

Voltage:
Time:
SDS concentration:

[bob1]

15 v
overnight.
0.1 % SDS

What size range is your protein/marker?  Most proteins don't blow through PVDF.

[protolder]

polaris, on 13 April 2011 - 01:28 PM, said:

I'm trying to use tris/glycine/20%MeOH (Towbin's) with 0.1% SDS to wet transfer onto PVDF. I think I'm blowing right through the membrane; my markers are not on the membrane or the gel after 100V for 2hrs.

If you use Towbin's buffer and a wet transfer system to PVDF (mine is bioRad mini):

What is your:

Voltage:
Time:
SDS concentration:

Hola, your conditions are adequates for transfer, so have you check polarity, and activate PVDF with methanol For me these are the causes to fail. Buena suerte

[polaris]

bob1, on 13 April 2011 - 03:35 PM, said:

15 v
overnight.
0.1 % SDS

What size range is your protein/marker?  Most proteins don't blow through PVDF.

Markers go from 14k to 97k.

[polaris]

[/quote]
Hola, your conditions are adequates for transfer, so have you check polarity, and activate PVDF with methanol For me these are the causes to fail. Buena suerte
[/quote]


Polarity is correct, transfer goes from black to red side, gel is on black side membrane is on red side, PVDF is activated and rinsed...

[knuf]

I use only 10% MeOH and no SDS, but I either transfer at 100 V for 2 hrs or 30 V overnight.

[mdfenko]

it is recommended that you use no more than 0.05% sds in the transfer buffer or it may not strip properly, thereby interfering with binding to the membrane.

check the western blot handbook.

Attached Files

•  Western blot handbook.pdf   2.03MB   320 downloads

Edited by mdfenko, 15 April 2011 - 11:41 AM.