Western blot problem....laddering pattern of white bands on Nitrocellulose membr - (Mar/25/2015 )

Hello everyone!

I am doing western blot to detect a 17 kDa protein with 0.22 um Nitrocellulose membrane. I am doing semi dry transfer with 20% ethanol in transfer buffer. Problem is I am getting white bands on membrane after I stain with DAB. Same is with Beta actin (white laddering of bands) Please find the attached photos. 

Details.

Transfer: Semi dry @ 50 mAmp for 2 1/2 hrs

Blocking : BSA 3% for 1 1/2 hrs

Primary : Goat polyclonal (1:500) overnight

Secondary: Bovine anti-goat (1:2500) for 1 1/2 hrs

For Beta actin:  Mouse monoclonal and anti-mouse HRP at 1:1000 and 1:2000

Western blot problem....laddering pattern of white bands on Nitrocellulose membrane ?. Available from: https://www.researchgate.net/post/Western_blot_problemladdering_pattern_of_white_bands_on_Nitrocellulose_membrane .

i don't know which brand semi-dry apparatus you're using but 2 1/2 hours is too long to transfer a 17 kDa protein (check the manual that came with the apparatus). with the semi-dry apparatus i've used, i wouldn't use more than 40 minutes, and that may be too long. you should determine how long you actually need to sufficiently transfer your protein of interest. the transfer time will also be affected by the acrylamide percentage of the gel.

what i think i see on your blots is membrane protected from developing background staining by the transferred protein and no binding of the secondary antibody (or the attached hrp is non-functional, it can be killed with azide).

you can test the hrp by adding some antibody to a dab substrate solution and seeing if you get the precipitate or by developing a spot blot of the antibody.

more information about your procedure is necessary for further evaluation.

I agree with you that 2 1/2 hrs are more than required, but as you can see the coomassie stained gel there are still bands in the lower part corresponding to small proteins. Also, you can see on the membrane there is no laddering or white bands on the lower membrane. Bands are only on the upper part of the membrane (High mwt proteins). To check whether if small proteins are blowing through the membrane I had kept another membrane below the first one, and even on that membrane laddering was on the upper part of the membrane only !!  That implies small proteins taking longer than the bigger one to come off the gel ?? 

Regarding HRP-linked secondary, I have been using anti-mouse HRP regularly , there is no problem with it ....though I am not sure about the anti-goat because I am using it for the first time. Also, if it was bad anti-goat HRP, beta actin should have showed up ?!

Further details: 15% gel

Primary incubated overnight; membrane has little dried up when checked next morning. I again pored in 2-3 ml of PBS-T and kept for further 1 hr on shaker.

i hadn't realized that the stained gel was after transfer, but that doesn't change most of what i said. the gel is overloaded so transfer was nowhere near complete for the higher mw proteins, another factor in retaining the high mw proteins is the restrictiveness of the 15% gel. a 10% gel would have been a good choice for separating your 17 kDa protein from actin, which is ~42 kDa.

it's possible, but not likely, that the 17 kDa protein blew through both membranes but, even then, i would expect actin to still be there. this is one of the reasons i think that your hrp has been inhibited. maybe there's some azide in the buffer(s) or maybe there's too much peroxide in the substrate solution (this can burn out the hrp) or there's no peroxide in the substrate solution (how old is the peroxide stock?).

Thanks for replying.

Why there are no bands on membrane for smaller proteins? Whilst you can see them on gel... If they have passed through the membrane! Also, as I said membrane was dried up next morning (primary incubation).. Could that be a reason?

drying nitrocellulose membranes while incubating can never be good.

it's possible that significant transfer didn't occur (hence the heavily stained gel after transfer) for some reason or other (electrical continuity, uneven contact with gel, ...). some capillary transfer may have occurred to give you the "ghost" bands.