western-blot: unspecific bands - How to reduce the amount of unspecific bands??? (Sep/01/2006 )

Hi,

I have a HA-tagged protein and I am using monoclonal antibody to detect it. The problem is that I get so many bands that is difficult to know which is the right one. Does anyone know any tricks to reduce the amount of unspecific bands??

Thank you!

Adriana

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what is ur blocking solution? and how long do u block ?

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For blocking I use 5% non-fat milk in TBST for 40min at RT.

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U could try blocking with 4% BSA. this may help.

or block with 5% milk for 2-3 hrs RT.

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Try using the antibody at higher dilution as well. I find I often have siimilar problems blotting for other tags, like GFP or FLAG and getting lots of background. I use my GFP antibody at 1:8,000 to get reasonable signal.

How much protein are you loading on the gel? More than say 50ug can increase background.

Also try blocking Overnight rather than just an hour if thats your protocol.

If you still get lots of background just be really careful and include a positive control if available.

Good Luck

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Hi,
Thank you for your advice.
I used the primary antibody at 1:2000 and second at 1:2500. Should I dilute only the secondary or both?
I am using only 20 µg of protein.

Adriana

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Hi,

I will do 2 gels (same samples) and I decided I will variate only the blocking agent.
So eather BSA or milk for 2 hours as you suggested.
I will also use more diluted antibodies (for both equally).
Let's see what I will get (next week of course )

have a nice weekend!

Adriana

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