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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 Optimization

Western Blot Optimization - Western Blot Forum

Western Blot Optimization - 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 01-15-2006, 08:46 PM
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Unhappy Western Blot Optimization



How can I optimize my western blots?
i have run several gels but i still cannot get a good film!
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  #2  
Old 01-15-2006, 08:50 PM
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Default Best way to optimize Western blots

The best way to optimize Western blots is to run 2 gels that are the same sample loaded in all lanes.
-Then you can transfer these to membrane.

- cut these membranes into 4s (be careful to cut between the lanes). mark the tops (this is required to know where the size markers are).

- optimize each cut piece differently but keep everything else the same change 1 thing at a time

- block with BSA for half, block with Milk 5% for the other half

- vary 10 primary antibody

- vary secondary a bit

- if signal is weak incubate with 10 in 4C overnight

More information on optimizing your Western Blot

hope this helps
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Last edited by admin; 12-23-2010 at 03:58 PM.
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  #3  
Old 08-10-2006, 08:00 PM
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Default Re: Western Blot Optimization

In most cases a high background signal is due to a secondary ab. Sometimes it has to be diluted as much as 1:100 000 - 1: 500 000.
Good luck
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Old 08-10-2006, 11:07 PM
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Default Re: Western Blot Optimization

Hey Marcin, Good idea also !
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Old 02-24-2007, 02:53 PM
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Default Re: Western Blot Optimization

Washing with generous amuntts of buffer 4-5 times after secondary really helps in minimizing the background.

Also when puring the wash buffer, dont pour directly over the membrane.

Also when you do the trial and error method, load just one sample with different protein levels.This helps in finding the optimum protein con. which has the elast number of non specific bands.
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Old 02-28-2007, 11:06 PM
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Default Re: Western Blot Optimization

What voltage does everyone run their gles at:

I run at 120V for 90 min. For transfer, I run at 100V for 90 min.

Do you run at different voltages depending n the size of your proteins?

Thanks
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  #7  
Old 10-16-2007, 04:20 AM
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Default Re: Western Blot Optimization

Hello Annopbal,
the best running of gels is usually at constant current I find.

If you are in a rush, 100V - 120V should be ok but not as sharp as constant current methods.

Also, if you want good gels stacking gels on top of your resolving are a must.

Cheers
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Old 12-02-2007, 04:56 PM
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Default Re: Western Blot Optimization

Hi,

I am new in the forum but so far I found it really useful.
We generally transfer the gel for an hour at 100V; we keep the transfer buffer in the fridge to avoid over heat and it could be use a couple of times.
After the transferring, I stained the gel with comassie staining, just to double check that most of the proteins are not any more in the gel.

Cheers
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Old 12-04-2007, 01:50 PM
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Default Re: Western Blot Optimization

Hi
same here. Please let me know how do you use comassive staining

Thanks
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Old 12-04-2007, 08:53 PM
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Default Re: Western Blot Optimization

Quote:
Originally Posted by rimapant View Post
Hi
same here. Please let me know how do you use comassive staining

Thanks
You know.....perform the western transfer and throw the used gel in staining solution to check transfer efficiency.
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