Hi Marcel,

It's been a while since I did SD transfer. However, as far as I remember It use to take less than 30 min for complete transfer.

In fact I routinely do wet transfer and it doesn't take more than an hour for complete transfer of even large sized proteins.

Ohh Ok, I saw now that you are using ETHANOL and not METHANOL in your buffer.

Just try 20% METHANOL in your buffer and it shouldn't take more than 30 min to complete transfer.

I also see that you use 0.08% SDS, I generally don't use SDS in my transfer buffer, however, the concentration that you are using shouldn't hurt.

Good luck.

S

Just to clarify, use METHANOL in place of ETHANOL in your buffer and keep the rest of the formula unchanged.

Dear Marcel

it really depends from the mw angr and kind of protein that you would like to transfer.

i'm using the i-blot (Thermo) which is able to transfer from gel to nc in 7 minutes and for my point of view work well with the most of proteins with me in the range 10-200KDa.

my opinion is that it is amazing and the. main problem is the cost (10-15 euro) each transfer, while other my colleagues do not like it and they continue to work in old style.

https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system.html

ciao

Manuele

I also use the iBlot system from Thermo. It is a dry transfer, except for a little water on a piece of filter paper. I love using it. The time it saves is worth the cost in my opinion, but might be too much for a university.

Lindsey Gallardo Manuele Martinelli

Thanks for the Info. The cost is exactly the problem. 10 Euro for one blot is a lot more than our regular consumables cost (maybe 2 - 3 Euro per Blot) Therfore, I was hoping for a cheaper method.

Sudeep Kumar

Thanks for the advice. But why does methanol allow for faster transfer than ethanol? Also MW-wise, everything from 10 to 150 kDa is relevant for us. Sometimes we want to do quantification of ubiquitinated species, meaning we need the HMW area with a good transfer.

And in general:

Can we just increase the current? In the beginning of the transfer, the voltage is very low (between 5 and 10 Volts).

I dont have any good explanation for this. Probably it has to do with fixation/ retention of your proteins.

I recently had this experience when I used Ethanol in place of methanol because we ran out of Methanol. Most proteins were not retained in the western blot.

My suggestion would be to just do a 20 min transfer of molecular weight markers and see how different MW proteins are transferred on Methanol or Ethanol buffer.

Probably 25V.

also check that there is no short circuit between the blotting papers that are used to sandwitch the gel and pvdf/nc membrane. the circuitry should only allow movement of ions through the gel to pvdf/nc.

My lab uses any blot pad and then the turbo transfer buffer in bulk. The individually packed sets are silly in my opinion. I use the Mixed MW protocol on the machine which is a higher V but for only 7 mins. I find this works great for >20ug per gel down to 15kda and up to 85kda. It may work for other sizes but I haven't looked.

336mM Tris, 260mM Glycine, 140mM Tricine and 2.4mM EDTA is a suitable rapid protein transfer buffer. Not necessary to pH. One 60 cm2 gel ~ 1.3A for 12-15 min.

For WB 10% ethanol in western blot transfer buffer works well and 100% ethanol for wetting the membrane.

Also dry or seminary transfer work well reducing waste. Or alcohol free transfer Dunn (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, with 50 µM SDS for large transmembrane proteins). Alcohol is more important for small proteins.