A big problem of my WB today - (Mar/26/2008 )

I used the following conditions:

5% skim-milk (Bio-Rad) in TBS+0.05%Tween 20, blocking at RT for 1 hr, then move to 4C overnight (no agitation, we don't have a shaker at 4C)

Primary Ab, 1:1000, in TBST with 2% skim milk, 2hr

Secondary Ab (anti-rabbit-IgG-AP, Sigma), 1:4000, in TBST with 2% skim milk, 1hr

Between each treatment I washed the membrane with TBST for 3 times, 10min each

Consequently I got a very high background. Nearly the whole membrane became purple after a few minutes of developing, although the bands on positive control were still visible.

I have to repeat it this friday. Can anyone help?

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In my experience best way to remove background is to do more washes.
I used to wash my membrane before blocking 2-3 times 5min each, after blocking, 2-3 times 5min each, and in between antibody incubations 5 times for 5 min each (instead of 3 times 10 min, is actually shorter ) also, not sure which developing system you are using (never heard of purple membranes before), but remember the detergent in your wash might interact with the substrate.

after my secondary antibody, and before developing I always wash 3-5 times with detergent followed by 2-3 times with TBS (NO Tween).

Also, I always used TBSTT= that's TBS 0.1% Triton-X100, 0.05% Tween 20.
hope this helps, good luck

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You might want to switch to an HRP-conjugated secondary if you have it available. I've never liked alkaline phosphatase myself - I think the membranes do tend to turn at least slightly purple with AP. Otherwise, you might want to eliminate the washes between the blocking and primary, increase your other washes - more washes, shorter incubation time (5 min).

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i would follow smu2's advice but i would also further dilute the antibodies (especially the secondary).

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More washes really helps! I washed my membrane 6 times 5 mins each. After developing, the membrane looks wonderful!

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I personally find that washing 5X for 5mins each in TBST works well, i always get nice clean blots.
Also the overnight step in primary is important for background, i don't do it at 4C but at RT on a roller, (and i find it doesnt degrade the proteins which contradicts what some protocols state)
AP does have its problems with background, if you could find an antibody with HRP you should find a lot of improvement.
I also wouldnt use any block in the diluent for my secondary antibody, just TBST.
If all else fails, try a secondary-only control, where omit the primary step and you probe with the secondary antibody and develop to check for background, this will tell you how well your dilution of the antibody works.

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agitation during incubation is a necessary at least to increase specific binding and decrease unpsecific binding;

think of alternatives to classical shaker, f.i. a shaking bath

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0.05%? Is it sufficient? The people in my lab using like 0.1 to 0.25% Tween. Is it normal? Does it has any effect regarding the background?

Thanks

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hye...i'm also hv the problem with my wb .i get the high background and i don't know how to solve..
for u'r information my protocol

blocking with starting block 1 hr
primart Ab incubation- overnight
wash-5x5 minutes
secondary Ab incubation - 1 hr
wash 5X5 minutes
Incubate with substrate 5 minute..
can anyon help me and wht can u say about my wb from the attach file
plssss..help me..
tq

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have you tried to re do your Western Blot? Or perhaps it is the detection problem. The bands are pretty weak. What method are you using for detection?

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