Problem with western blot detection- strong background and weak signal

Hi,

I m trying to double confirm the western blot results i got from my polyclonal antibody are specific by repeating it with a monoclonal antibody.

However my bands are weak and kind of blended into the strong background.

I loaded a total of 30ug of crude lysate, overnight incubation with 0.6ug/ml of primary antibody (mouse anti-human), 1 hr of goat anti-mouse AP (biorad), and 400ul of immunosubstrate (biorad). Can't see anything at 5, 10 and 30 mins. Over-develop till 2hrs , but background becomes very strong.

All washes are 10mins x3 in 1% TBST, the antibodies are in 1% TBST with 5% non-fat milk.

I was thinking of loading up to 150ug-200ug ( i could lyse more cells in a smaller volume of lysis buffer) of crude lysate during the SDS-PAGE, would that be too excessive for the lane ?

Help !!!

1. Is you monoclonal antibody specific to your protein? I mean made from whatever tissue you are working on? if not, I will trust the polyclonal and assume that the monoclonal does not crossact

2. and if you used the same loading for your previous experiment, I do not think increase loading will be wise. 150-200ug protein per line will be too too crowded and won't separate well on minigel

3. are you sure you are using 1% TBST, usually people use 0.1%

4. for blocking reagent, BSA is suggested

Ops sorry, typo, i mean 0.1% TBST.

The monoclonal antibody is specific, my lab mate managed to make an elisa using the antibody.

I will try to use the BSA as blocking reagent, around 5% ?

Erm, what would the maximum loading capacity for each lane on a 15 well minigel ?

about the loading capacity, it is not suggested for >50ug per lane on a 15well 0.75mm minigel

according to my experience, 2% BSA will be enough for blocking

it seems that your 1st antibody is already pretty concentrated, but sometimes commercial antibodies are not as concentrated as they say in their manual. so maybe try also double that concentration.

and for AP detection, lumigen (www.lumigen.com) has very sensitive chemiluminescence reagent.

What is your detection method? If you are using ECL, there are more sensitive substrates you can use (SuperSignal, etc.). People tend to have the false assumption that higher the sensitivity of the subtrate, the higher the background signal; however, this is not the case since these substrates (when used as directed) should be employed in conjunction with lower amounts of primary/secondary antibody, which decreeases background while the more sensitive substrate increases the signal.