Immunoblotting - traditional method
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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