Western Blot Optimization

[dave2006]

How can I optimize my western blots?
i have run several gels but i still cannot get a good film!

[admin]

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

hope this helps
Molecular station admin

[marcin]

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

[kiki06]

Hey Marcin, Good idea also !

[anoopbal]

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.

[anoopbal]

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

[admin]

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

[marcomonopoli]

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

[rimapant]

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

Thanks

[danfive]

Originally Posted by rimapant

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.