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Non-specific bands with secondary Antibody on Western Blotting - (Nov/10/2008 )

Hi guys n gals,
I am having trouble with non-specific bands with my goat anti-mouse secondary antibody in the region of 65-30 KDa. Proteins of my interest are in 43,32,30, 26 and 22 KDa. Its is getting difficult to interpret the bands due to the non-sp bands. My protocol is as follows:
1) Run my gel at 90V for about 11/2- 2hrs
2) Wet transfer at 100V for 75 min at 4 deg celcius ( I keep the apparatus in the freeze). The markers seem transferred well with this setting. I dry my membrane after rinse in PBS before-
3) Blocking in 10% NF milk and 5% Goat Nromal Serum at RT for 2 hrs-- three 15 min washes in TBS-t after rinse.
4) Mouse monoclonal Ab at dil of 1:1000 at 4degrees overnight on roller in 3%milk
5) Three 10 min washes in TBS-T
6) Second block in 3% Human serum albumin (HSA) at RT for 1-2 hr ( this is because my boss thought that the cross reactivity of the goat Ab to human proteins in my samples my be delt with human serum albumin- but this has not helped).
7) Secondary at dil 1:10,000 in 3% milk + 3% Goat n Serum in TBS-T at RT for 1 hour on roller
8) three 20 min washes in copious amounts of TBS-T
9) Read with Gene snap Chemi box using Millipore ECL reagents. I incubate the membt=rane in the ECL reagents ( also I do ensure that the ECL reagents are at room temp before use) and take images at 1-3-5 min exposures.
Is my primary too concentrated? Does my secondary need diluting-- I have tried 1:20,000 with little help.
I have also incubated my secondary in 3% Milk+3% HsA at 1:20,000 for 45 min with no luck.
Any suggestions will be greatly appreciated.


hello lazeleo
please change your protocol to this shortened one:
1)blocking is done for 1hr at room temperature in 3% skim milk dissolved in TBST.
2) after blocking no need to wash in TBST and proceed directly with the primary Ab (either at room temperature for 1 hr or onvernight at 4 degrees)
3) after primary Ab, wash 3 times 3% skim milk dissolved in TBST (5 min each)
4) incubate with secondary Ab for 1 hr at room temperature then was 3 times with TBST (5 min each) then 2 times in TBS (5 min each)
5) develop the membrane
As for the non specific bands, they mainly depend on 2 things, your primary Ab type (whethr its mono or polyclonal) and its source and the cell line you are using. Blocking methods usually can not eliminate cross reactivity to those bands.