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Special Western Blot Troubles - help neede (Jun/06/2007 )

I have continued problems with my western blot. DonĀ“t know what this means. (a typical amidoblack staining)
Can anyone out there help me, or is able to tell me why this phenomenon occurs so often (mostly when I drive 2 transfers in parallel)

Thanks

-moljul-

looks like might be some type of a bubble in the transfer? gel melting because of heat? maybe you aren't as careful when doing two gels or it is too warm with two?? clean your gel and transfer boxes really well and make up new solutions, make sure you transfer on a mixing plate with a stirbar and ice pack...

HTH and good luck!

-beccaf22-

[quote name='beccaf22' date='Jun 6 2007, 07:38 AM' post='100678']
looks like might be some type of a bubble in the transfer?

I also think it looks like a bubble of air in the transfer. When you put your membrane on top of your gel make sure there are no airpockets in between the two - get something cylindrical like a tube and roll out any possible air bubbles. Auldmok

-auldmok-

I be sure that this phenomenon is not due to air-inclusion. I remove all air through rolling with a pipette. There is also no heat induction, because I always use pre-cooled transfer buffer, ice and ice packagings. And always take a look that the voltage is not too high. I think this is not a banal failure.

-moljul-

QUOTE (moljul @ Jun 6 2007, 07:46 PM)
I be sure that this phenomenon is not due to air-inclusion. I remove all air through rolling with a pipette. There is also no heat induction, because I always use pre-cooled transfer buffer, ice and ice packagings. And always take a look that the voltage is not too high. I think this is not a banal failure.


hmm, most of the membrane seems to be quite normal and okay. maybe you can use another blotting device and maybe try another membrane? i think it is not an airbubble, there is a slight staining visible or not. looks like an incomplte transfer or something.

-Lab Dance-

I had the same thing happen to me, it's not an air bubble... What I did was add some more wattmann papers to make sure the membrane is as close to the gel as possible. Check the membrane before, leave it in transfer buffer before you use it and make sure it has no white spots on it. And although you use ice and cooling, it still may be heating up, what I did was reduce the voltage and increase transfer time and that solved my problem. Good luck

-syaniv-

QUOTE (syaniv @ Jun 13 2007, 02:28 PM)
I had the same thing happen to me, it's not an air bubble... What I did was add some more wattmann papers to make sure the membrane is as close to the gel as possible. Check the membrane before, leave it in transfer buffer before you use it and make sure it has no white spots on it. And although you use ice and cooling, it still may be heating up, what I did was reduce the voltage and increase transfer time and that solved my problem. Good luck


Thank you for your tips. With which conditions you drive your transfer (I normally use 225-250mA)?

-moljul-

Hey,

The person above who noted that it is not a bubble but used more Whatmans is on the right track
I have seen this numerous times. It is the result of an incomplete/inefficient transfer.

To fix it:
#1 USe 4 pieces of Whatman paper for each transfer. Two per side.
#2 Only run 1 (IMPT!!!!!!!!) transfer per tank. It is rare in my lab that two transfers run in parallel in the
same tank will work.
#3 We always run transfers overnight at 35 volts (~70 miliamps).

Do the transfers this way and you will never see those large empty patches on your membranes again.

-mikew-

The voltage/amp I use depends on the gel. I use mini-gel by bio-rad, or midi and maxi gels by hoefer so I can't tell you specifically. The midi hoefer is transferred at 150V for about 2h (depending on protein size) with the apparatus placed in ice. The bio rad is basically the same. If I can be of more help, let me know. Also tell me if it worked!

-syaniv-