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Poll: Time and voltage for wet transfer to PVDF


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6 replies to this topic

#1 polaris

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Posted 13 April 2011 - 01:28 PM

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:

#2 bob1

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Posted 13 April 2011 - 03:35 PM

15 v
overnight.
0.1 % SDS

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

#3 protolder

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Posted 13 April 2011 - 10:17 PM

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

#4 polaris

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Posted 14 April 2011 - 07:06 AM

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.

#5 polaris

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Posted 14 April 2011 - 07:44 AM

[/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...

#6 knuf

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Posted 14 April 2011 - 09:44 AM

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

#7 mdfenko

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Posted 15 April 2011 - 11:38 AM

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.

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

talent does what it can
genius does what it must
i do what i get paid to do




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