Missing patches on immunoblot - please help! (View forum version)



sciencerage

Posted 15 February 2011 - 05:16 PM

Hi everyone. I am suddenly experiencing a problem with a WB I have done succesfully several times before. I have not changed my protocol but am now seeing blank patches when I develop the blots - I have posted an attachment showing the worst case. I also included the ponceau stain to show that this is not a protein transfer issue (such as a bubble between the gel and membrane). I do primary incubations on a rocker overnight at 4C, and secondary incubations on a shaker for 1hr at RT. I do not allow the membrane to dry out and also ensure that the ECL substrate covers the entire membrane. I stripped/reprobed the membrane in the attachement and saw the same missing patches, so it is unlikely that the problem has to do with uneven distribution of any reagents. I also don't think it is because the rollers in the developer are dirty because no one else is having this problem.

Any advice would be greatly appreciated. Thanks.


mdfenko

Posted 16 February 2011 - 11:25 AM

how and where do you pour your wash solutions onto the membrane? the clear area looks a bit like a splatter pattern.

sciencerage

Posted 16 February 2011 - 01:33 PM

I don't pour solutions directly onto the membrane; my wash solutions are poured into a container with multiple compartments, then I transfer the membrane (using forceps) into different compartments for each wash. The exception would be the ECL reagent - I pipet that directly onto the membrane and roll it around to get full coverage. I agree it looks like a splatter, but I can't imagine of what.

knuf

Posted 17 February 2011 - 06:34 AM

To me it kind of looks like maybe you didn't have enough antibody mixture to cover the membrane completely--so when it went on the shaker platform the outer edges got more antibody and have a more uniform appearance that the inner bands. Don't know if that's possible for your setup, but thats what it looks like.

mdfenko

Posted 17 February 2011 - 01:42 PM

during the exposure with the ecl reagents, was the cassette perfectly flat? is the cassette rigid or soft (cardboard)? was something placed on top of the cassette during exposure?

sciencerage

Posted 17 February 2011 - 02:49 PM

during the exposure with the ecl reagents, was the cassette perfectly flat? is the cassette rigid or soft (cardboard)? was something placed on top of the cassette during exposure?


I do the ECL reagent exposure with the membrane on the countertop so it's flat. Then I let the reagent run off and place the membrane in the cassette, which is rigid, and cover the membrane with clean transparency film, which I tape down to hold in place. Nothing is placed on the cassette during exposure - I am usually doing fairly short exposures so I just hold the film on top of the membrane.

mdfenko

Posted 18 February 2011 - 07:30 AM

have you tried a different lot of film?

lab5

Posted 19 February 2011 - 03:27 PM

Hi guys,

we had similar problem once or twice, seen this you should wash the membrane and reapply ECL taking care of even coverage of the blot completelly and with enough ECL, if it slips away reapply with the pipette all the incubation time with the EL do not leave the blot, if this doesn't help the problem is earlier in the process which means antibody incubation, for that phase we had before some very very hydofobic membrane and had uneven bands, what we start doing is flipping the blot in the container with the side for antibody binding on the bottom of the container and it made it certain that antibody si bond equally throughout the surface, worked.

lab5