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Western Blot Wet Transfer Conditions


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#1 steinpdh

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Posted 21 May 2014 - 12:23 PM

I have a (rather general) question concerning the wet transfer conditions of a western blot. Transfer conditions obviously vary widely depending on many things like transfer buffer formulation, acrylamide concentration and thickness of the gel, size and pI of protein of interest, membrane material etc. and that they ultimately have to be optimized for every single application.

 

But, when looking through general guidelines of manufacturers I have come across a rather puzzling discrepancy. All details aside, for general wet transfer of "regular" Tris-Glycine gels Life Technologies recommends a constant voltage setting of 25 V for 1-2 h, whereas Bio-Rad recommends constant 30 V for 16 h (or a "high-intensity" setting of 100 V for 1 h).

 

25 V for 1-2h vs. 30 V for 16h (or 100 V for 1 h)

 

How is this obvious discrepancy explained? I am confused huh.png



#2 bob1

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Posted 21 May 2014 - 01:49 PM

25 V for 1-2 hours will transfer smallish proteins fairly effectively (up to about 50 kDa), and transferring at 30 V overnight (16 h or so), or even using a lower Voltage such as 15 V will effectively transfer all your proteins, regardless of the size (I know this works up to about 400 kDa at least).  The main risk with the long transfer is transferring the small proteins through the membrane rather than on to it.

 

100 V for 1 h will blow any small proteins lower than about 20 kDa through your membrane (though this can be ameliorated by using certain types of membrane, such as small pore PVDF).  100 V will transfer largish proteins OK, up to about 150 kDa, larger than this will struggle to come across.  I would only use these conditions if I had an abundant protein in the 30-150 kDa range.



#3 steinpdh

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Posted 21 May 2014 - 02:16 PM

Thanks for your answer! It gives a nice overview. What I also take from it is that the size of the protein of interest is ultimately the most important parameter? What is more critical for the transfer of high pI proteins though? pH of the transfer buffer or presence (in gel) or addition (to transfer buffer) of SDS?

 

Let's say my protein of interest is ~ 24 kDa and my main goal is to get complete transfer out of the gel, no blowing through the membrane and the sharpest possible bands. Which condition would be more favourable, a slow one like 15 V overnight or something faster like 60 V for 1-2 h?



#4 bob1

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Posted 21 May 2014 - 02:23 PM

I always go for long and slow - I usually use 15-20 V overnight with 20% methanol.  The methanol strips the SDS from the protein, and impedes its transfer somewhat as it shrinks the pores of the gel (also prevents swelling so gives you sharper bands)

 

I don't know about transfer of high pI proteins, but I think you would probably want to go with a different buffering system with a different pH to Towbin's original recipe.



#5 steinpdh

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Posted 21 May 2014 - 02:37 PM

I have read somewhere that if you don't equilibrate the gel in the transfer buffer the leftover SDS in the gel will be sufficient for proper transfer of higher pI proteins out of the gel. When hitting the membrane the methanol strips off the remaining SDS and increases binding efficiency. Does that make sense? But might this actually decrease the resolution since the methanol meanwhile shrinks the gel during transfer?



#6 bob1

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Posted 22 May 2014 - 01:23 AM

It could be that not equilibrating would work.  During equilibration the gel actually expands - the methanol helps prevent this, so there shouldn't be loss of resolution, especially once the gel is in the sandwich and under slight pressure.



#7 mdfenko

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Posted 22 May 2014 - 04:05 AM

also, keep in mind that the presence of sds makes the pI of the protein a less significant factor in migration out of the gel (until the sds is stripped off by the methanol).


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#8 steinpdh

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Posted 23 May 2014 - 12:38 AM

True, that is what I meant. So how would a Towbin buffer with 0.1 % SDS work, as SDS and methanol kind of play opposing roles?

 

Would it maybe be a good idea to equilibrate the gel in Towbin buffer containing 0.1 % SDS (instead of omitting equilibration after SDS-PAGE) but then transfer in regular Towbin buffer without SDS?

 

And one other question. Life Technologies recommends using a half-strength Towbin buffer with only 12 mM Tris and 96 mM Glycine. Why?

 

Thanks for all the help guys :D



#9 mdfenko

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Posted 23 May 2014 - 04:03 AM

towbin buffer with sds (i, and millipore, recommend to use no more than 0.05%, some say 0.1% but that may be too much to properly strip) should work just fine.

 

equilibration prior to transfer is recommended but you should still use towbin + sds or the sds will be stripped off too early (with methanol and sds you'll have a dynamic process)

 

reducing buffer concentration changes its conductivity. this will alter the electronic characteristics of the transfer. you can try it if you are not satisfied with the results under the conditions we discussed, but, you know the old adage "if it ain't broke, don't fix it".


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#10 steinpdh

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Posted 23 May 2014 - 05:20 AM

Will try a transfer at 15 V over night with Towbin buffer containing 0.05 % SDS next time.

 

See, the funny thing is, I am supposed to transfer cardiac Troponin I which is actually supposed to have a pI of 9.87. But the antibody manufacturer's protocol and virtually every publication that states its conditions for transfer of cTnI uses regular Towbin. I haven't had any luck with Towbin yet though, but neither with CAPS at pH 11. When using Towbin I have also tried to put a second membrane on the cathode side of the gel, but the tiny little bit of cTnI that did come out of the gel was actually on the anode side. That's what puzzles me considering its pI of 9.87.

 

There should be a database for best knowledge western blot transfer conditions for every protein.



#11 mdfenko

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Posted 27 May 2014 - 04:36 AM

pI is unimportant when the protein is denatured with sds (a bit of a simplification). however, when sds is stripped off the pI becomes important again.

 

you may find this handbook from millipore helpful.


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