Blank spots on WB after semi-dry, NO BUBBLES - (Mar/12/2013 )
I get random blank spots on my blot which sometimes overlap my protein of interest. I have made CERTAIN that the membrane is not dry, the filter paper is wet, and that there are no bubbles. I use a roller to make sure no bubbles are present and by eye there appears to be no bubbles, yet I still get these spots. Does anyone have any ideas?
I transfer at 100 mA for 40 minutes for a mini-gel in a Fisher semi-dry blot unit. I'm slightly weighing the top of the blotter down with an ice bucket since the sandwich is too thick to tighen the knobs.
Where there are no blank spots - the transfer looks great.
Thanks!
Mike
Do you have another rig you can try? Are you sure you are getting even current flow across your whole apparatus (If you test your rig with a multimeter, is it showing the correct amperage)? Your gel is running properly? If you ponceau your blot and you see a big circle, it has be from 1)air bubbles, 2)faulty equipment (but would faulty current transfer produce a distint bubble pattern - probably not).
Only advice I have is try another labs semi-dry rig or reduce the amount of watmans you use in your sandwhich. You should be able to fully close the appartus, you should not place an ice bucket on top. This is going to interfere with you electrodes forming a closed circuit.
The gel runs great and I only get the distortion occasionally - not every blot, so I don't think it is the equipment. Would too much filter paper cause bubbles? Like I said - when I'm putting it together I make sure there are absolutely no bubbles present. Also - I thought the extra weight on top would actually improve the current transfer since everything is forced in to close contact. Thank you for your help!
it would seem that you are getting bubble formation between the gel and membrane during the transfer.
you may need to degas your transfer buffers prior to wetting the filter papers and soaking the gel.
it could be that your filter papers are too wet.
it could be from insufficient removal of sds from the gel during the pre-transfer soak.
it could be happening because of insufficient pressure on the sandwich.
why are your sandwiches too thick to properly close the apparatus?