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Does anyone know an effective buffer mix to use for high current Western transfers? We are successfully using the vendor's premixed buffer to transfer a wide range of protein sizes to PVDF membranes at 1A/25V for 10 min. We get great results with the vendor's expensive buffer.

I haven't been able to find a non-proprietary recipe that works. The best ones we have hold the 25V, but then have a decrease in conductivity (increase in resistance) where they will start at 1A, but drop to 0.4A by the end of the 10 min. The vendor's buffer seems to hold the high current and results in better transfers.

I don't know if it will be helpful to list all the variations I've tried in detail, but they have revolved around modifying a traditional Towbin buffer plus SDS, more glycine/Tris, or MgCl2. These were all various suggestions from people around the department, but I haven't found much published evidence for a buffer under these conditions.

I realize 1A (and no I don't mean 1mA) is a lot of current, but the vendor's system works really well. Any pointers on what I might try/add would be appreciated even if you don't have a worked out protocol.

Edit: Info from the MSDS indicates it has 3 reagents:

Listing of dangerous and non-hazardous components:
Proprietary Reagent K
10-20%
Proprietary Reagent EB II
5-10%
Proprietary Reagent S
1.0-2.5%
7732-18-5 water
50-100%
·
.....
Solvent content:
Organic solvents:
0.0 %
Water:
74.8 %
Solids content:
25.2 %

[I'm not sure if this MSDS info should be put here, just because I'm looking for someone who has already used a high current buffer they know the formulation for, not a guess to what I have.]

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I can't help you with respect to wet transfer buffers. If you don't mind using proprietary systems, I highly recommend the iBlot system (Invitrogen). It's a dry transfer, available in nitrocellulose or PVDF membranes, and takes ~7 minutes for a complete transfer. It may seem expensive, but I bet it will save you money in the long run. –  leonardo Jul 14 '13 at 3:29
    
@leonardo I believe that even though Life Technologies (gotta keep up with the mergers) would call iBlot a "dry transfer," that it really seems like a semi-dry transfer with very nice pre-packaged cell set up. I have used them, and the gels certainly seemed wetter than dry. I feel like Licor as more of a 3-rd party non-player had an interesting analysis. –  Atl LED Jul 14 '13 at 4:06
    
Yes, Life Tech is now the parent company. ;) The analysis is interesting, but I would conclude from the report that iBlot has similar performance to wet and simi-dry transfer methods. The biggest improvement I've found was the greater resolution and sharpness of bands over manually poured SDS-acrylamide gels. –  leonardo Jul 14 '13 at 13:13
    
@leonardo although you get the same resolution/sharpness using precast gels and the standard Towbin's wet transfer method... –  MattDMo Jul 14 '13 at 15:31
1  
@AtlLED - I understand where you're coming from, I was just trying to clarify leonardo's comment about clarity. FWIW, I used to run at least that many westerns a day: I used Invitrogen precast gels, and did a wet transfer at 100V (constant) for 1hr@4C. One of the issues we found with the iBlot was its transfer efficiency of very small/large proteins - you can get one, or the other, but generally not both. –  MattDMo Jul 15 '13 at 14:39

2 Answers 2

up vote 1 down vote accepted

So after a lot of work in optimization, I thought I would post what worked best for me. This buffer recipe was able to successfully transfer EGFR and insulin from the same lysate, and a clear band for both (large and small protein respectively). 10% SDS-PAGE gels were transferred at 1A for 10 min.

High Current Transfer Buffer

  1. 48 mM Tris
  2. 15 mM HEPPS
  3. 1.0 mM EDTA
  4. 1.3 mM NaHSO3
  5. 1.3 mM N,N-dimethylformamide
  6. 25 Mm gLY-GLY
  7. 20% Methanol (v/v)

I do hope this can help someone else, and want to site Garic et al as wonderful starting point. Their publication was made after I asked the question, as @user4148 pointed out.

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The SB buffer has been around for quite a while, and I definitely have used it successfully before for both agarose and PAGE gel buffers for some time. The only thing is that you have to run the gel in a cold room (high voltage equals high temperature) and that you have to optimize the timing and conditions so that your samples don't run off the gel.

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I've used SB. First, isn't it a running buffer, not a transfer buffer (or at least I've never heard of it being used in transfer), second I believe the formulation is still some what broad no? –  Atl LED Jul 16 '13 at 23:17
    
If you look at the running buffer recipe it contains SDS, whereas the transfer buffer contains methanol. Using the same base buffer but adding the different reagent, I have used it before. However, you might have to try it with your specific recipe and running conditions to optimize it. –  Michael Lai Jul 16 '13 at 23:29
    
A recipe with SB buffer for western mcdb.ucla.edu/Research/Banerjee/protocols/Western-Blotting.pdf –  Michael Lai Jul 16 '13 at 23:34
    
My point wasn't that I couldn't find SB recipes, my point was I could find several. I will give the linked one a try and see how it stands to optimization. I still can't find examples of SB in high current low voltage situations (especially for protein transfer). The one you linked notes 25V o/n, which is the opposite of what I'm trying for. –  Atl LED Jul 16 '13 at 23:46
    
I am just pointing out that it can be used for transfer buffers as well. But interesting enough people seem to have mixed experiences with it. I think in general you probably don't want to run fast transfers if you are looking for very precise or accurate results because it is generally a trade-off that you need to consider. –  Michael Lai Jul 16 '13 at 23:55

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