Why do I have to almost "burn" my transfer before I can detect?? - (Oct/21/2009 )
Well, I finally bought Inmobillon Psq, and still the same problem. This time I tried on the semi-wet system (xcellII blot module) 30V for 30 mins as the tech support of Invitrogen suggested and nothing!!!!! The loading marker perfectly transferred though. I'll increase the time to see what happens.
I will try this buffer next time!!
medchemgirl on Oct 22 2009, 02:19 PM said:
K.B. on Oct 22 2009, 04:36 AM said:
I suspect it's something other than the transfer...
Do you know you have a significant amount of protein in the gel? Have you tried staining the membrane (if it's there by staining but not by your HRP protocol, then it's your detection method and not the transfer that's screwing you up)? Try putting a piece of membrane on the other side of the gel as well; maybe your protein is oppositely charged at the pH of transfer..
Did the low molecular weight portion of the ladder transfer correctly?
You could also try 20% MeOH in the transfer buffer, and/or adding some extra SDS...
Well, I haven't stained the gel after SDS, but I have use a standard protein, I load from 5 to 25 ug of pure protein, and I have nice blots with it, only when I let the system dry, otherwise I don't see anything. From my real samples I load 50 to 100ug of sample.
HomeBrew on Nov 11 2009, 08:07 PM said:
Do you know you have a significant amount of protein in the gel? Have you tried staining the membrane (if it's there by staining but not by your HRP protocol, then it's your detection method and not the transfer that's screwing you up)? Try putting a piece of membrane on the other side of the gel as well; maybe your protein is oppositely charged at the pH of transfer..
Did the low molecular weight portion of the ladder transfer correctly?
You could also try 20% MeOH in the transfer buffer, and/or adding some extra SDS...
Is that like a 100V? My system uses volts.
K.B. on Oct 22 2009, 04:36 AM said:
Ok, I used the Dunn's carbonate buffer and it really didn't go well.
For some reason the gel did not hold and started to melt, and the protein ladder looked all screwed up on the membrane.
medchemgirl on Nov 12 2009, 05:37 PM said:
K.B. on Oct 22 2009, 04:36 AM said:
No, it's actually quite low - approx. 10~15V.
medchemgirl on Oct 21 2009, 11:35 AM said:
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.
Well,this is because heat impairs the gel structure and allows proteins to flow through smoothly. To achieve even transfer temperature should be over 65ºC
Ok, now it makes sense, but anyways, the gel almost melted. I used 20V though.
K.B. on Nov 13 2009, 12:26 PM said:
medchemgirl on Nov 19 2009, 12:20 PM said:
K.B. on Nov 13 2009, 12:26 PM said:
Yep, when heated it looks almost melted
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