WB trasfer problem - (Sep/19/2007 )

hello eveyone

i am having an annoying problem lately and i dont know what to do , i got air bubbles in my western blot film??

i tried everything , check the thickness of my sponges, put my PVDF membrane in methanol then water then one hour in blotting

buffer, put my gel 15 minutes in blotting buffer before starting blotting,get rid of air bubbles that appear during making the blot

sandwitch by forceps very gently and pour some buffer on it.........REALLY GOING CRAZY, i have to repeat almost evey wb i

make ???????

and buffer compostion is fine we share it and no one else got this problem soo frequent??

any advice?
thankx

do you monitor the current and voltage during the transfer? maybe the wattage is going too high and you're overheating the blot.

check to see if the transfer is getting too hot and adjust the cooling and/or current, if necessary.

Thankx mdfenko

but i am using the same electrical suuply for gel electrophoresis and its ok ?

I assume you roll something over the blot just to make sure the bubble have all come out.


I would make the transfer sandwich submerged in the buffer and then roll something over the filter and sponge to make sure there are no bubbles.

Hi,

Air bubble on film? X-ray film?
And not during the dismantling of the gel and PVDF membrane? If you can't see bubbles when you dismantle the gel and PVDF, then it might probably be that your PVDF membrane was dried out during transfer. As mdfenko suggest, careful not to overheat during transfer (can try placing the blotting apparatus into ice-box and surround it with ice during transfer). This will cool down the rising temperature during transfer.

If PVDF is dried out after transfer, you could re-wet it with methanol for 1 to 5 min (or until membrane is 'wet'). Then wash with H2O, 5' before immunodetection.

...-...

scolix i am doing as you said and still no use
i am running (all blue ) marker with my samples thats why i can see some air bubbles after transfer on the (all blue )bands and sometimes the marker got transfered but still got air bubbles after developing the film, by the way its like a whole area missing from the film or sometimes a spot inside the band

about heating i am using my buffer ice cold and i put ice cold water outside the blot and i didnt chnage any step from what i used to do a year ago so\????/
could the electrical power suuply goes wrong all of a sudden?

it wouldn't necessarily be a problem with the power supply. it could be a problem with the transfer buffer. are you sure it was made the same way as in the past? did someone else prepare it? did you use a new lot of one or more component?

hi medfenko
yes i am sure about the transfer buffer as its a common buffer and no one else got the same problem...

i changed the power supply unit today and still got the same problem.....

going crazy really!!!!!

a question ? can we put 2 membrane in one plastic container for methanol soaking and therafter buffer soaking ? or should be separated?