2 replies to this topic

[fotolilith]

After a seemingly normal running phase of my western blot, I transfered 2 minigels overnight at 33v & room temp and 20 hours later opened the sandwiches to discover that both membranes were blank and the molecular markers were gone.

The western blot running phase seemed okay - my lab ran out of running buffer, so the supply company said we could use the transfer buffer instead with an additional amount of sds - we ran a practice gel with this, which worked very well.

Specifically, in this problem run I used nitrocellulose membrane, and one gel was loaded with 72 ug, while the other was loaded with 3 ug of protein in each well.

I stained both membranes with coomassie blue, and while one seemed to retain striking amounts of protein, the other seemed completely empty (so I really did not know what to make of it).

Can anyone tell me why my molecular marker dyes would disappear during a 20 hour transfer at such a low voltage? I've transfered overnight before with no similar problems.

Could the proteins have blown through?

Could the anode and cathode have been switched when the apparatus was loaded?

Could the apparatus have gotten too hot overnight?

Could the molecular markers have just traveled faster (and through) the membrane?

Could there have been some problem with the buffer or gels (both were 10% acrylamide).

Am I likely to have any protein left on the membrane (I'm probing for a low molecular weight protein on one membrane and a high molecular weight on the other).

Thanks for your help!

Edited by fotolilith, 29 July 2004 - 08:06 PM.

[Sprag]

You do not specify whether it's wet- or semi-wet- transfer. But yes, the protein can easily go through your membrane!! You could try to stain with Ponceau to see if there's any protein on your membrane, but if your marker is gone, I would doubt it.

SDS in the transfer do change the transfering of larger proteins, as more SDS binds to proteins and therefore make them more negative charged and easier to transfer. The larger the protein, the more SDS and the more negative charged. But the SDS is not necessary in your transfer buffer.

My transfer buffer is :

25 mM Tris pH 9.4
40 mM Glycine
10% methanol

And transfer at 35V for 55min in Semidry transfer (Invitrogen)

If you do overnight, put it in the coldroom.

[wirly]

Doh!  Your poles were switched.  
Done this a couple times myself.  Normaly you'd see no protein, but in a double transfer one will pick up some protein though on ONE membrane it will prebably be really difuse.  Dyed markers may not show up at all.