7 replies to this topic

[Prep!]

Hey all.. i have been using PVDF all my life... but now we are out of stock and have a NC membrane..
i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!

[almost a doctor]

Pradeep Iyer, on Nov 6 2009, 04:24 AM, said:

Hey all.. i have been using PVDF all my life... but now we are out of stock and have a NC membrane..
i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!

Hi, you dont need to remove the methanol from the transfer buffer, in fact not sure whether it would work without methanol.

Also remember that even if you dont need to pre-wet in 100% methanol, you still need to pre-wet your NC membrane in transfer buffer (containing methanol) before assembling the gel-membrane sandwich.

[Prep!]

yup... point noted...
thanx a ton!!

[mdfenko]

methanol is not required for activation of nitrocellulose, but it has the same purpose in the transfer buffer as with pvdf (stripping sds from protein, preventing gel swelling).

for almost a doctor, the transfer will work without methanol. transfer from non-denaturing gels can be done without methanol (you can use buffer without methanol with sds-page but the transfer efficiency will be reduced), especially if you don't use sds in the transfer buffer. but, why prepare two buffers and confuse everybody.

[almost a doctor]

mdfenko, on Nov 6 2009, 05:09 PM, said:

methanol is not required for activation of nitrocellulose, but it has the same purpose in the transfer buffer as with pvdf (stripping sds from protein, preventing gel swelling).

for almost a doctor, the transfer will work without methanol. transfer from non-denaturing gels can be done without methanol (you can use buffer without methanol with sds-page but the transfer efficiency will be reduced), especially if you don't use sds in the transfer buffer. but, why prepare two buffers and confuse everybody.

thanks mdfenko!!  I always enjoy learning with your posts

[Feelcontraire]

Pradeep Iyer, on Nov 5 2009, 08:24 PM, said:

Hey all.. i have been using PVDF all my life... but now we are out of stock and have a NC membrane..
i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!

I may be wrong but I think I remember that at least 10% metanol was requiered in transfer buffer for nitrocelulose, not the case of PVDF.

[mdfenko]

Feelcontraire, on Nov 18 2009, 08:42 PM, said:

Pradeep Iyer, on Nov 5 2009, 08:24 PM, said:

Hey all.. i have been using PVDF all my life... but now we are out of stock and have a NC membrane..
i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!

I may be wrong but I think I remember that at least 10% methanol was required in transfer buffer for nitrocellulose, not the case of PVDF.

methanol is only required in the transfer to strip sds from the protein being transferred and to help prevent (or reduce) swelling of the gel during the transfer.

it has no direct effect on the nc (it does not require "activation" like pvdf). you can transfer without using methanol at all (with non-denaturing gels).

[Feelcontraire]

mdfenko, on Nov 19 2009, 10:28 AM, said:

Feelcontraire, on Nov 18 2009, 08:42 PM, said:

Pradeep Iyer, on Nov 5 2009, 08:24 PM, said:

Hey all.. i have been using PVDF all my life... but now we are out of stock and have a NC membrane..
i was wondering if i can follow the same protocol for blotting as i do with PVDF.. except for the activation with methanol part of course...
but my transfer buffer also has 20% methanol.. can i continue using tat or remove it from my buffer for a NC??!!!
i usually run at 100V and 400mA max current for an hour!!!

I may be wrong but I think I remember that at least 10% methanol was required in transfer buffer for nitrocellulose, not the case of PVDF.

methanol is only required in the transfer to strip sds from the protein being transferred and to help prevent (or reduce) swelling of the gel during the transfer.

it has no direct effect on the nc (it does not require "activation" like pvdf). you can transfer without using methanol at all (with non-denaturing gels).

Yep, probably it has to be with the fact that nc has less retention capabilities for some proteins and that it is not activated, so methanol is plain zero. I read somewhere that 1% or less may be more than enough for transfers, but I haven't tried that.