Immunoblotting - traditional method

 

rehydrate western in PBS-Tween for 5 min

add primary antibody in a minimal amount of PBS-Tween and incubate on a rocking platform for 1 hr min (alternatively overnight at 4C with the tray wrapped in Saran Wrap)

rinse western twice in PBS-Tween for 5 min

add secondary antibody in a minimal amount of PBS-Tween and incubate on a rocking platform for 1 hr

rinse western twice in PBS-Tween for 5 min

rinse western once in pH 9.5 Buffer for 5 min

during this rinse prepare the nBT-BCIP Alkaline Phosphatase Color Development Reagent

decant pH 9.5 Buffer and add color development reagent (immediately after it is prepared)

color development will occur for about 5 min and should be stopped before the background increases significantly

decant color development reagent and rinse twice quickly with PBS-Tween

wash for 5 min in PBS-Tween plus EDTA (100 ul of 0.5 M stock per 10 ml)

dry blot on paper towels - blot needs to be kept in the dark to prevent the color from fading (store between sheets of blotting paper)

Immunoblotting with Chemiluminescence Reagents

use the same procedure as above but use the appropriate secondary antibody for the chemiluminescent reagent used

add secondary antibody in a minimal amount of PBS-Tween and incubate on a rocking platform for 1 hr

rinse western three times in PBS-Tween for 5 min

use blotting paper to remove excess PBS-Tween from the blot

mix the chemiluminescent reagent and add to the membrane

if the blot is too wet the reagent will diffuse and create excessive background

expose to X-ray film immediately, the most intense signal is observed in the first few minutes

Reprobing westerns

westerns probed with chemiluminescent reagents can be stripped of antibodies and reprobed

Western Stripping Buffer (pH 6.8)

SDS   2 g/100 ml

Tris-HCL 1 g/100 ml

prepare the above solution, adjust the pH, and make to 100 ml

then add the B-mercaptoethanol (in the hood)

BME  780 ul/100 ml

incubate the membrane for 30 min or longer at 50 C in Western Stripping Buffer

then wash the membrane three times in PBS-Tween for 5 min

the blot is then ready to be reprobed