Getting a SDS-page like picture in a PVDF blot??!!

[Lee Yean]

Hi everyone,

I am new in WB and hope to get some helps from you guys in optimizing my runs. Thanks.

I am getting a SDS-page like picture in a PVDF blot, which is very unusual. Please have a look of the attached files. Do you have any idea on what went wrong with the runs? The ladder is a prestain ladder so it shouldn't be detected by a chemiluminescene system but I got it in my gel picture which is weird to me. I have tried to increase the washing time and tween buffer concentration in the wash buffer in order to reduce the background signal but it doesn't really work.

Following is the information of the runs.
1. Membrane used = PVDF membrane (pretreat with methanol for a few seconds before soak it in transfer buffer)
2. Detection system used = GAM HRP Detection System
3. Primary Antibody Dilution: 1:2,500
4. Secondary Antibodry Dilution 1:10,000
5. Expected protein size: 92 kDa
6. Transfer method: Semi-dry transfer with 48mA for 1 hr 45 mins.

Thanks again.

Best wishes,
Lee Yean

[pigfarmer]

I often see Ladder bands in my westerns as well. I think it may have something to do with leaving too much of the chemiluminescent on the membrane...not really a washing problem. Also, longer exposure times seem to give this result as well.

I do not think it is a problem though, if you look at the 'bands' they are really absense of background luminescence, not bands at all.

[Pippuri]

Hi Lee Yean,

What kind of blocking condition you are using?

I suggest you try to use different kind of blocking condition, also, try to dilute your primary antibody to a lower concentration....

Good luck.

[Lee Yean]

Hi Pigfarmer,
Thanks for your comment. But the bands were detected within the range of my expected protein size. So, I think there must be something I did wrong to get this reverse image (negative staining result). I'll decrease my exposure time on my next run, thanks :-)

Hi Pippuri,
I blocked my membrane after transfered using 3% BSA for 50mins at Room temperature. My supervisor also recommended me to use 5% skim milk powder, think I'll give it a try. thanks.

yeah, I need lots of luck now ... :-)

[Lee Yean]

Hi All,

I got the bands I wanted after changing my blocking reagent from 3% BSA to 5% skim milk. The explanation from my assoc. supervisor was as follow. FYI.

" Looking at the result the most likely reason I can think is not blocking your membrane. If for instance by mistake you used buffer not containing the blocking protein, then in the next step your primary antibody would non-specifically bind to available sites all over the membrane and when you come in with the secondary antibody it will bind on the primary and any free sites still available thus you will get the dark background. The only places you will not get reaction is where your proteins already occupy the sites thus the negative staining."

cheers,
Lee Yean