Why do I have to almost "burn" my transfer before I can detect?? - (Oct/21/2009 )

Hi,
I am completely puzzled by this. Every time I try to transfer a particular protein, it wont stick to the membrane unless I run it to the point where the buffer evaporates, almost like baking the system. I am using the semi-dry blotting system from Invitrogen. Even if I use the same amount of voltage (50V, Invitrogen protocol calls for 20V) and keep it really cold that it doesnt evaporates, I won't see my protein, unless I let it go until the buffer evaporates and it smells like burnt! I don't get it! is my protein that hard to to stick? its a 17KDa protein, very small!
I have done this multiple times as to determine that the only way I can detect it is by frying the membrane!

Any comments please?? I'm about to call Invitrogen and ask about this.

What kind of membrane are you using? What is the composition of your transfer buffer? What kind of gel are you transferring from? How are you detecting the protein?

Sorry,
I am using NT (Biotrace from VWR), I'm using the Invitrogen NuPAGE transfer buffer (10%MetOH), NuPAGE Novex Bis-Tris gel. I'm using HRP as 2ry with ECL detection.

HomeBrew on Oct 21 2009, 02:37 PM said:

I had problems with the NuPAGE systems, so now I use Biorad Tris-HCl gels and make my own transfer buffer:

3.03 g Tris base
200 ml methanol
500 ml H2O
adjust pH to 8.3
add 14.4 g glycine
q.s. to 1 L

Isn't the max voltage for a SD 15V? (I use BioRad.)

We use the NuPage system as well, and the NuPAGE transfer buffer -- we used to make it up with 20% MeOH, but now we use 20% EtOH -- less disposal problems, less toxicity, and it works just as well. The real difference is we use 20% alcohol, whereas you're using 10%. I've heard spiking it with some SDS can help the transfer of difficult proteins, but we haven't had to try it. We're using the XCell SureLock Mini-Cell apparatus with PVDF membranes.

I get very nice transfers of low MW proteins (as low as 6-8 kDa) with Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20% MeOH) and Immobilon Psq from Millipore (PVDF, 0.22 um) on semi-dry system for 1 hour at 0,8 mA per cm2 of membrane.

Thanks a lot!
The max voltage for the Invitrogen Xcell SureLock is 20V, but it wasn't working with 20V so I had to crank it up to 50V accidentally evaporating the inner chamber buffer and luckily getting my transfer to work! I've repeated it several times with same results, if I don't do that, everything else transfer except my protein. I proved it with the standard protein as well.

lab rat on Oct 21 2009, 05:44 PM said:

That Immobilon membrane sounded good until I saw the price!! haha.

K.B. on Oct 22 2009, 04:36 AM said:

medchemgirl on Oct 22 2009, 03:19 PM said:

We use 0.22 um PVDF membrane/filter paper sandwiches from Invitrogen. With price, it's relative -- if you've done the experiment multiple times, and haven't got the data or image you need, and it would have worked the first time using PVDF, have you really saved anything?

"if you've done the experiment multiple times, and haven't got the data or image you need, and it would have worked the first time using PVDF, have you really saved anything?"

Ditto the SD blotter if it gets damaged by too-high voltage.