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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Western Blot - Extra Bands

Western Blot - Extra Bands - Western Blot Forum

Western Blot - Extra Bands - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 07-28-2006, 08:39 PM
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Question Western Blot - Extra Bands



Hey guys,
I have a western blot with too many extra bands. I have tried using BSA and milk during primary and secondary antibody incubations. I still get too many bands...

what gives?!

can someone help me with this,
thanks
Moleculardude
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Old 07-30-2006, 06:25 AM
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Default Re: Western Blot - Extra Bands

Do you think it is your antibody?

Are you using fresh samples?
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Old 08-10-2006, 07:54 PM
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Default Re: Western Blot - Extra Bands

Try to dilute your primary or/and secondary solution. What is your current concentration?

You can also block your blot overnight at 4C instead of 1-2 hrs at rt.

If bands are lower than your target protein, they can be degradation products. Make sure you handle your samples on ice and use proper protease inhibitors.

If bands are higher, they can be dimers or other oligomers of your protein. This problem can be solved e.g. by adding urea to the loading buffer.

Good luck!
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Old 10-17-2006, 03:24 PM
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Default Re: Western Blot - Extra Bands

It maight be unespecific binding of your secondary antibody. Lets say your tissues are from sheep/goat and your secondary antibody is a Rabbit/Mouse/Human anti Goat IgG it could be that your secondary antibody is binding to your first one and to other IgGs in the sample. To solve this problem you have to change the secondary antibody and consequently the first ... meaning, your first can not be raised in a host similar to the species you are using.
Hope this helps
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