Hi Friends,

I am lysing the QT6 cells transfected after 48 hrs and doing my SDS-PAGE and western blotting. My protein has the HA antigen tag and i use 1:1000 dilution of HA monoclonal Ab.

To troubleshoot this problem v 2 people tried with the same blot, the same procedure and the same buffers. But i am getting again with high background. But the other person result was with clear band (only my protein of interst) without any bg.

It is difficult to identify where things r going wrong with my handling.
brief steps:
1) 1 hr blocking with 5% skim milk prepared in 1X TBS-T(tween 20 0.05%)
----after this, mild wash with 1X PBS-T to remove traces of skim milk.
2) 1 hr primary ab(1:1000) prepared in 1X TBS-T
3) wash 3 times with 10 min duration each with 1X TBS-T
4) 1 hr Sec ab (1:1000) prepared in 1X TBS-T
5) wash 3 times with 10 min duration each, with 1X TBS-T
6) ECL -Amersham Reagent and developing.

Expecting a reply
raje.

If the other person(s) is getting ok results while you are not; work with the other person and split the procedure between the two of you. You can do steps 1, 3, 5 and the other person can take steps 2, 4 and 6. In a parallel experiment, switch and do steps 2,4,6 yourself while the other person takes over steps 1, 3, 5. This is the easiest way to troubleshoot experiments that work in the hand of one person but does not in the hands of another.

Myself I never wash aways skim milk. I use 1% skim milk in TBST for bloking and then add primary antibodies. That reduces background.

In western blotting, all the steps are important.

I agree that milk in antibody solution can reduce the background.
But I think that the washing steps are important too.
Do you do a rapid wash with 1X PBS-T to remove traces of antibody after incubation each antibody incubations ?
It's really important to remove it....

You ought to wash in 30 or 50 ml ? is the wash volume enough ??

Try to wash one or two more time and see the result !

David.

Seems you're following standard procedures.

Here are couple of things that may help.

Clean your staining vessels really well. Maybe there are residual proteins about.

Use a clear sheet protector (cut off the three ring side to make a flap) rather than Saran wrap in your film cassette.

I use 2ndarys around 1:2500 or 1:4000.

Try 3% IgG free BSA instead of milk.

Filter or centrifuge your milk blocking solution.

If these simple things don't work, maybe load less protein or check that proteins are transferring well.

Good luck

HI, i had quite similar problems when i was working with my BXP-21 monoclonal antibody. I was not vortexing it after taking it from the freezer. My mentor told me that a good biochemist always mixes things. Anyway, vortex all of your stuff before applying to the membrane. See you...

Hi there,
I am also having big troubles with my westerns, here are the tips I have learned from this troubleshooting period :
- the dilution of the primary antibody is crucial regarding the background
- the incubation with the primary antibody should be either around 4 hours at room temp or overnight at 4 degrees.
- the most important with the washes is not their duration (5 mn is very good) I recommand a quick wash prior to the main sequence of washes to remove promptly the excedent of antibody straight away. The volume used for the washes can make a great difference also (the more the better). the incubation with the secondary antibody should not exceed 1 hour at room temp.
If you have any more advises...

Good luck

Julien

I had suffered aimilar problem.
Afetr transfering, I wash the membrane twice to remove some debris.
It improved my result!

You do everything right. Just a few small things I'd like to recommend:
- use 5% skimmed milk as dilution solution for antibodies (primary and secondary);
- use TBST for all washings except for the last one. After incubation with the secondary Abs make 3 washes with TBST and then one more with TBS.

Hi,
The staining container/vessel DO matter when it comes to background! Make sure you have washed it well ie. with lab detergent and final rinse with deionized water and drying it in a clean tray. Or next time round, use a new clean container. Hope that helps

Ally

High background and non-specific bands can be reduced by washing in a TBS buffer supplemented with 0.5M NaCl and 0.2% SDS. Wash for 10-30 minutes. This may also reduce the binding to your target antigen so your mileage may vary.

For phospho-proteins, use BSA instead of dry milk. Casein in milk is a phospho-protein and causes high background.

Hi Raje,

Try incubating membrane with primary ab (anti HA) 1:2000 for 1 hr (which I follow for this ab specifically).
hope this will help u.

Nath.

1:1000 is an extremely high concentration for a secondary antibody! This would definately give you high background. We use our secondarys at 1:10,000 to 1:25,000 dilutions - but it depends on your specific seconday of course!

try once again...may be the problem was during the process only...so once again follow the same process and then see the results..

I think one problem I had was incubating overnight in milk buffer without shaking. Initially my protocol was either to block for 1 hour at room temperature with shaking, or block overnight without shaking at 4*C. Then I stopped blocking overnight, and instead used methanol to soak the newly transferred blot, followed by air-drying, followed by storing the blot between tissues overnight.

We had been used to seeing a lot of random spots on the background of the blot upon visualizing -- but when it was dried out overnight instead of soaking in milk solution without shaking, most of these background spots were gone.