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 reagents:

            a) Fluorescence = 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. 

            b) Chemiluminescence = CPD*

 

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.

 

Wash once with detection buffer

 

Fluorescence Detection

 

            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.

 

Chemiluminescence Detection

 

            Place blot face up on one sheet in an open sheet protector

            Add ~ 1ml reagent to blot, spread covering the blot with other sheet

            Incubate 5 minutes at room temp

            Expose to film

 

For 2006, we need to make:

 

2 Liters of 10X TBS

 

4 Liters of Blot Wash

 

I have everything else ready to go.