Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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I recently started having problems transferring Western blots (12%gel --> nitrocellulose) - when I retrieve the blot, the ladder is missing and all the protein has vanished. The first time it happened, I had two blots in the tank - one was fine, the other was blank. I tried immunoblotting it just to see if it was a problem with the ladder, but there was nothing in any of the lanes. Last night I had only one blot in the tank, and it came out blank. I always transfer at 30V at 4degC overnight and have never had a problem until now. The bottles of 10x transfer buffer and methanol were the same ones that I had been successfully using before. I tried adding extra layers of whatman paper to make sure the sandwich is pressed tight, which didn't help. When I start the voltage I can see the bubbles coming off of the outside wires from under the plastic tubing, the current seems to be normal (about .15Amps, which my post-doc friend says is what she always finds when she checks), and I can't see any wires disconnected or corroded. In short, I can't come up with a single thing that's changed between when it worked and when it didn't. The only thing I can think of is to try another tank, but since I can't find anything wrong with this one, I'm not sure if that will work. Any advice?
well if your protein is vanishing, then the most probable thing is that the current is wrong. it's either too high (and the protein goes through the membrane) or too low (and the protein is still in the gel - though in this case you should still be able to see the marker in the gel). I would just use another power source and see if that works. Alternatively, you can transfer for half the time or something like that and see if you get any protein.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
Well the simplest fix is that you are putting the cassette or gel to nitrocellulose in backwards. When you do this, the protein is pushed away from the nitrocellulose and there will be absolutely nothing including the ladder on the nitrocellulose membrane. Two ways to remember this is black to black meaning put the cassette black side to the black side of the box. The second is run to red- meaning the gel will transfer to the nitrocellulose towards the red side of the box (black side of box-whatman-gel-nitrocellulose-whatman-red side of box)
current runs this way >>>>>>>>>>>>>>>>>>>>>>>>>>>
The foolproof way to determine whether the box is deficient or if the orientation is wrong simply put a nitrocellulose membrane on either side of the gel. If there is something wrong with the box, then nothing will show up. However, if the orientation was backwards one of the nitrocellulose membranes will have your markers and protein. Try it. The other variable here is the transfer buffer. Is anyone else using it? If someone else is using it also, but their transfers are working, then its not likely the cause of your problems.
Another thing you can do to see if you have protein is stain with Ponceau S.
I look forward to hearing your results.
There was definitely a problem with the current; I think it just wasn't "getting through" (so to speak) and the protein was either diffusing away overnight or staying in the gel (which I didn't check, although since the markers were nowhere to be found I guessed that the protein had diffused out). It wasn't backwards - otherwise I would have seen the markers on the whatman paper. Luckily that's one mistake I've never managed to make!
There was some white salty buildup coating the wires running across the black side of the box - I cleaned it off and ran a test transfer, and it looks like things are back to normal. I can only guess that the residue was somehow interfering with the field running across to the red side so that, even though the current was reading normally it wasn't creating an effective field.
Thanks for all the input!
Check the transfer buffer ingredients too, I had this transfer problem once when I forgot to add SDS in transfer buffer. I was transferring two 12% SDS-PAGE gels to PVDF in one tank. 100V, 1 hr. One gel transferred fine, another went backwards to blot paper after 1 hr. Did I just arrange backwards? Did lack of SDS mess with electric gradient? Correlation may not be causation, so I don't claim lack of SDS was the problem. It would be easy to do an experiment, though.
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