Western Blotting and Immunostaining Protocol

Wash XCell box, blotting module and trays with soap and water before proceeding.

Reagents:

Transfer buffer

25 mM Tris HCl,

190 mM Glycine

10% Methanol

Blocking buffer

5% non-fat dry milk in TBS-TWEEN 20

or

3% BSA in TBS.

TBS-TWEEN 20

25 mM Tris HCl

137 mM NaCl

0.05% TWEEN-20

 Vectastain ABC kit

Species specific

 Substrate solution (4-chloro 1-napthol in methanol)

Dissolve 10 mg in 10 ml methanol just before use

30% Hydrogen Peroxide

Electro-Blotting Procedure:

Run SDS-PAGE as appropriate for protein of interest.  Meanwhile:

Soak blotting pads in transfer buffer, pressing down to remove trapped air bubbles.  Leave soaked pads in buffer until ready to assemble the blotting sandwich.

If using PVDF, pre-wet the membrane in pure methanol, then equilibrate in transfer buffer by shaking in a shallow tray filled with buffer.  Nitrocellulose can be placed directly into transfer buffer to equilibrate.

Note:  nitrocellulose membrane will dissolve in pure methanol.

Place the cathode core (the larger piece) of the Blot Module in the shallow glass tray half-filled with transfer buffer.  Place two soaked blotting pads in the cathode core, followed by one piece of blotting paper.  Check the buffer level in the tray at this point; it should just barely cover the blotting paper.

Carefully remove the electrophoresed gel from the plastic cassette; remove the bottom ridge and the wells at top by trimming with the gel knife.  Lay the gel on the pad-blotting paper assembly in the cathode core.  Using a wet (gloved!) finger, gently smooth away trapped air between the gel and blotting paper.

Wet the surface of the gel by gently squirting transfer buffer using a plastic transfer pipet.  Place the soaked blotting membrane on the gel, gently smoothing away trapped air between the gel and membrane.  If necessary, reposition the membrane once.

Working quickly, place a soaked piece of blotting paper on top of the membrane, followed by 3 to 4 soaked blotting pads.  The last pad should be about 1 cm above the edge of the cathode core.  Place the anode core on top, then carefully transfer the entire assembly to the XCell box, pressing the anode core into the cathode core with the wedge.
Set the power supply to deliver 30V and run for 1 hour.  Larger proteins may require longer transfer times. 

At the end of the run, remove the blotting membrane from the sandwich assembly.  Do not allow the membrane to dry before placing in 15 mls of blocking reagent.  Gently agitate for 1 hour before proceeding with immunostaining.

 Optional:

・    The gel can be stained after transfer as a check for untransferred protein.

・    The blotted membrane can be rinsed in water and dried for later analysis. 

Store between two pieces of dry filter paper, wrapped in plastic at 4°C.

  

Immunostaining Procedure:

All steps at room temperature unless otherwise specified.

After blocking the membrane for 1 hour, discard the blocking solution and add 15 mls of primary antibody solution diluted as appropriate in fresh blocking buffer.  Incubate for 1 hour.

Filter (0.22μm) the primary antibody solution into a 50ml tube, and store at 4°C for next use.  Wash the membrane with 3 x 15 ml of TBS.  Discard washes.

Add 15 mls of secondary antibody diluted in blocking buffer (use 1 drop per 15 mls) to the membrane and incubate for 1 hour with gentle agitation.

 Discard secondary antibody solution.  Wash the membrane with 3 x 15 ml of TBS.  Discard washes.

 Add 10 mls of TBS containing 2 drops each of reagent A and reagent B from the Vectastain kit to the membrane.  Incubate for 30 minutes with gentle agitation.

Discard the TBS-AB solution.  Wash the membrane with 3 x 15 ml of TBS.  Discard washes.

 Add 10 mls of TBS to the membrane.  Add 60ul of hydrogen peroxide followed quickly by the substrate solution.  Mix by gentle agitation.  It may take as long as 5 minutes to see color development.  Stop the reaction by removing the substrate mixture and rinsing several times with Milli-Q water.  When the reaction mixture is rinsed away, the membrane can be air-dried.  Store protected from moisture and light for preservation of color.

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