Mirabito’s BIO 510 Western Blot Probing and Detection Protocol
Reagents:
TBS = 200mM NaCl in 10 mM Tris-HCl pH7.4
10x TBS (per liter) : 72.4 g Tris Base, 118.6 g NaCl, pH7.4 with concentrated HCl
TBS0.5 = TBS with 17.5 g NaCl added to give a final [NaCl] of 0.5 mM
TBS0.5T = TBS0.5 with 0.1% Tween 20 (5 ml of a 20% stock per liter)
Blocking Solution = TBS0.5 containing 5% nonfat dry milk and 0.2 Tween 20
Antibody Diluent = TBS containing 1% BSA, 0.02% sodium azide
Western Blot Wash Buffer is TBS0.5T
For HRP-linked secondary antibodies, Antibody Diluent as above without sodium azide
Novagen Mouse Anti-His Monoclonal Antibody: dilution = 1:2000
Jackson Immunological Alkaline Phosphatase-linked Goat Anti-Mouse Antibody:
dilution = 1:10,000
Detection reagent = 1:1000 dilution of DDAO Phosphate Stock Solution (1.25 mg/ml in
dH2O) using 10 mM Tris-HCl pH 9.5, 1 mM MgCl2 as the diluent.
Protocol
Destain Ponseau-stained blots, block either 1 hr at room temp or overnight at 4 degrees.
Wash bloking solution with Western Blot Wash, shake off all wash buffer.
Probe blot in 10 ml of diluted primary antibody for 1 hour at 37 degrees
Wash three times with blot wash. A “wash” means, remove all the previous solution,
cover membrane with wash solution, throw that away, add at least 20 ml wash solution,
incubated with mixing for 5 minutes.
Remove all the blot wash, rinse, remove the rinse, shake off all wash buffer, add 10 ml
diluted secondary antibody. Incbuate for 1 hour at 37 degrees.
Wash three time with blot wash.
Add 10 ml detection reagent and incubate at 37 degrees for 10 minutes. Remove blot
from solution (but save the solution), place blot face up in a plastic sheet protector, and
image using the Typhoon as indicated below.
Set Typhoon for fluorescence. Choose fluorescence set up. Choose the 670BP30
emission filter and the 633nm laser. Set the PMT to 400.
If image is dim or blank, put filter back in detection reagent and incubate for up to 1 hour
at 37 degrees. If the image is very dark, repeat scan reducing the PMT settings as
necessary.
For 2006, we need to make:
2 Liters of 10X TBS
4 Liters of Blot Wash
I have everything else ready to go.