Homogenizing buffer for mouse liver tissue - (Dec/12/2008 )

hi,

the previous lab tech said to use 1x PBS + protease inhibitor cocktail as the homogenizing buffer for the mouse liver tissue for down stream western blotting. I was wondering whether using PBS for the homogenization step will interfere with AP linked secondary Ab?

general protocol steps:
- homogenize (PBS + protease inhibitor)
- protein assay with bradford reagent
- loading 40mg protein on 10% SDS-PAGE
- 3x wash with transfer buffer
- transfer onto nitrocellulouse membrane
- ponceau s stain of membrane
- NaOH wash (remove ponceau)
- 3x dH2O wash
- 2x TBST wash
- block with 5% milk in TBST
- 3x TBST wash
- primary Ab 2hrs
- 3x TBST
- secondary Ab-AP 2hrs
- 3x TBST
- 1ml Western lightning CPD-star ~3-5mins

I havent been able to get my westerns to work with this protocol from previously homogenized samples. even positive control with actin Ab didnt produce any luminescence with CDP-star.

anyway, my assumption was that the washing steps will remove any PBS from the homogenization step so it will not interfere with AP conjugated Ab correct?

thx.

running the sample on the gel removes the pbs.

ponceau washes off with water, why use naoh (which can hurt the proteins bound to the nitrocellulose) and what concentration?

I used water to rinse the membrane, and destain to visualize the protein bands, do i just keep rinsing with water to wash away ponceau?

The reason I used NaOH is b/c both the manufacture protocol and previous tech's notes says to use 0.1M NaOH to remove ponceau s prior to blocking.

cheers.
e

yes. just keep rinsing with water. keep in mind what hsp70 wrote, any remaining ponceau will come off when you block.