8 replies to this topic

[floyd78]

Hi all,

Hoping you can help me troubleshoot my problems with transferring ~220kDa proteins, I scoured these forums yesterday trying to pick up relevant tips but I don't seem to have cracked it yet.

5% acrylamide SDS containing minigels (10x10) loaded with 50ug protein per lane and run @ 200V for 45 mins, 2 checked with coomassie and confirmed clean, crisp bands and even loading. Markers up to 460kDa separated nicely.

2 gels put into wet transfer overnight in Towbin containing 5% methanol and 0.1% SDS with Millipore 0.45um pore PVDF @ 90V in coldroom. Previously tried transfer at 50V and the larger proteins stayed put in the gel.

This morning I disassembled and the buffer was fairly warm (not a great surprise given the higher voltage) but there was no sign of the prestained markers on either the gel or the membrane or on the filter paper. I was always under the impression that it is quite difficult for proteins to become unbound from PVDF and pass through the other side, is this not the case? I am just in the process of staining the gel to see if there is anything still left on it.

Any ideas about where I may have gone wrong?

Thanks in advance

Edited by floyd78, 14 October 2009 - 02:10 AM.

[mdfenko]

you may want to try less sds and more methanol.

sds added to facilitate transfer of large proteins should not exceed 0.05%. sds will interfere with the binding of the protein to the membrane (it will move too fast to be captured by pvdf, in nitrocellulose it will also be repelled by the negative charge).

10-20% methanol should be used with a buffer containing sds to strip the sds from the protein after it has exited the gel.

[Dr Teeth]

mdfenko, on Oct 14 2009, 12:45 PM, said:

you may want to try less sds and more methanol.

sds added to facilitate transfer of large proteins should not exceed 0.05%. sds will interfere with the binding of the protein to the membrane (it will move too fast to be captured by pvdf, in nitrocellulose it will also be repelled by the negative charge).

10-20% methanol should be used with a buffer containing sds to strip the sds from the protein after it has exited the gel.

Agree. I use 4-12% bis-tris gels and use a tris-glycine transfer buffer with 20% methanol and 0.05% sds and wet transfer overnight at 25 V. I routinely look at p300 and other high molecular weight proteins without trouble; however, also note that, for me, transferring at 45 V for 3 hours or various semidry transfers all failed to transfer proteins above ~150 kD very well .

[floyd78]

Thats great, thanks very much both

[clow]

Hi there,

I am trying to transfer a protein that is 500kDa and was wondering what conditions (voltage and time?) would be best for the wet transfer step.

I have tried semi-dry transfers with success (3 hours 25v), however the results are not reproducible (seems to be issues with heat and transfer efficiency when running such a long semi-dry transfer).

Dr. Teeth you mentioned 25v overnight, was that for your 300kDa protein.

I am also using a 20% methanol, 0.05% SDS transfer buffer.

Thank you,
Chris

[ANP]

Some have tried omitting both SDS and methanol in transfer buffer (25mM Tris, 192 mM glycine) and instead heated the buffer to 70-75 oC during transfer (40V for 10-20 min). Heat should "open" the gel and release proteins. Nitrocellulose must be soaked for 10 min in buffer before transfer (not required with methanol).

Kurien and Scofield 2009, Methods in Mol Biol (536).
Good luck

[Feelcontraire]

floyd78, on Oct 14 2009, 02:09 AM, said:

Hi all,

Hoping you can help me troubleshoot my problems with transferring ~220kDa proteins, I scoured these forums yesterday trying to pick up relevant tips but I don't seem to have cracked it yet.

5% acrylamide SDS containing minigels (10x10) loaded with 50ug protein per lane and run @ 200V for 45 mins, 2 checked with coomassie and confirmed clean, crisp bands and even loading. Markers up to 460kDa separated nicely.

2 gels put into wet transfer overnight in Towbin containing 5% methanol and 0.1% SDS with Millipore 0.45um pore PVDF @ 90V in coldroom. Previously tried transfer at 50V and the larger proteins stayed put in the gel.

This morning I disassembled and the buffer was fairly warm (not a great surprise given the higher voltage) but there was no sign of the prestained markers on either the gel or the membrane or on the filter paper. I was always under the impression that it is quite difficult for proteins to become unbound from PVDF and pass through the other side, is this not the case? I am just in the process of staining the gel to see if there is anything still left on it.

Any ideas about where I may have gone wrong?

Thanks in advance

Following the 1h 1amp rule, 90v OvN may be too much.

As you've been told you need SDS too accelerate the protein and Ethanol to stop it. Also heat really improves transfer, and over 65º makes it even.

I developed a system that works like a charm an is as follow (tank transfer).

sponge.m-paper.m-membrane-gel-paper.g-sponge.g

Activate the membrane in ethanol

Equilibrate in tris-gly buffer without sds and without ethanol everything but sponge.m.

Equilibrate sponge.m. in the same buffer but containing 20% ethanol.

I don't use SDS in this equilibration buffer as it makes bubles.

As transfer buffer use the tris gli but in this case without ethanol and with 0,1%SDS.


The high SDS will accelerate your proteins and the ethanol will flow from the sponge to the membrane removing the SDS and making the proteins stop there.

Avoid equilibrating the membrane in ethanol containing buffer as it will contact the gel and impair protein trip.

I usually heat the transfer buffer to 75º in the microwave and transfer for 30-60min-, 400mA.

If you are afraid the your protein surpases the first membrane ad a second membrane between paper.m and membrane.

Also if your protein has high IP it will trip to the (-)pole. Usually SDS will avoid this but if you want to be sure simply place another membrane between the gel and paper.g.

If your protein is travelling the wrong way you can use higher pH transfer buffer like CAPS ph 11.

This is inspired in a semidry blotting system but works as-well.

[mdfenko]

you use 0.1% sds in the transfer buffer?

the literature recommends to not exceed 0.05% sds in the transfer buffer, the methanol (or ethanol, in your case) will not be able to remove enough sds to ensure good capture by the membrane.

but, if it works for you then why change?

[Feelcontraire]

mdfenko, on Nov 19 2009, 10:18 AM, said:

you use 0.1% sds in the transfer buffer?

the literature recommends to not exceed 0.05% sds in the transfer buffer, the methanol (or ethanol, in your case) will not be able to remove enough sds to ensure good capture by the membrane.

but, if it works for you then why change?

Well, that recommendations comes from the fact that everything will be soaked in SDS-Methanol containing buffer but in this case the methanol containing layer doesn't have SDS(of course it will flow in during transfer).

I think that the semidry transfer system also uses 0,1% on one side (I may be wrong) I even read of some very efficient transfer with up to 0,5% SDS.