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Almost (?) accidental use of 10X transfer buffer for 350-kDa protein


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#1 Hic5Prostate

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Posted 11 August 2014 - 01:36 PM

So it's been a while since I've done wet transfer (in my graduate lab, we used semi-dry).  I'm trying to transfer ATM, a 350-kDa protein, to a PVDF membrane from a 6% Tris-Glycine gel.  When I took my apparatus to the cold room to begin the transfer (but before I hooked it up), I noticed that precipitate was forming in the transfer buffer.  It was at that point that I realized that I had accidentally made my buffer with 10X Tris-Glycine and 20% methanol, not with 1X Tris-Glycine and methanol.  I immediately took it back into the lab, where I set up another apparatus with 1X buffer + methanol and transferred the entire cassette from the first box.  I then set up everything else as normal and started the overnight transfer.

 

So, the big question is whether I messed with the integrity of my experiment.  Does exposing the gel to higher concentrations of Tris-Glycine cause any adverse effects to the proteins in the gel, such as leeching?  I have a feeling that I'm in the clear, because I realized what I did prior to starting the transfer, but I just want to make sure.



#2 mdfenko

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Posted 12 August 2014 - 03:59 AM

whether there's an effect on the transfer depends on how well you drained the apparatus and sandwich. if not well then ionic strength of your transfer buffer may be on the high side. that may affect the transfer.

 

otherwise, it should be okay. but you should remember that transfer starts as soon as the gel comes in contact with the membrane.

 

as for leaching, as long as the gel is in medium (this includes the equilibration step in semi-dry transfer) and there is no current you will experience some diffusion. that's why you want to apply the current as soon after introducing the sandwich to the apparatus as possible.

 

ps- you can facilitate transfer of high molecular weight proteins by adding up to 0.05% sds to the transfer buffer.


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#3 Hic5Prostate

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Posted 12 August 2014 - 04:45 AM

I don't think the sandwiches (there were two) could have been drained because of soaking. All I did was move the electrode with the sandwiches into a new container. So I guess that means the ionic strength might be on the high side. I guess I'll have to check the Ponceau staining when I take a look at it.

#4 mdfenko

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Posted 12 August 2014 - 05:22 AM

when you pulled the sandwich out of the apparatus, it probably drained sufficiently to ameliorate any effects of the high ionic strength medium.

 

as you said, you can check with ponceau.


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#5 Hic5Prostate

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Posted 12 August 2014 - 10:59 AM

Thx for the reply.  The standards did transfer.  However, I did not see my desired band at 350 kDa in the Ponceau stain (and because the lysate for the IP was in RIPA, I didn't see anything else).  I will take your suggestion on using the added SDS to the buffer and optimize the condition.






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