Western Blotting Protocol

Material and Equipment:

PVDF is used for protein sequencing, kept wet and pre-soaked in MeOH and more endurable for repetitive usage.

Or Nitrocellulose (1 pc.) 7.5 x 8.5cm

Filter paper (4 pcs.) 7.5 x 8.5cm

Flat-end tweezers

Western Blot Transfer Cell

Small container for the blot

Plastic bag sealer and sealable bags

Transparencies (2 pcs.)

Dupont NEN oxidizing and luminol reagent

Solutions:

TBS: 50mM Tris, pH 7.6 12.12g / 2L

150mM NaCl 17.52g / 2L

TBS/Tween: TBS + 0.05% Tween-20 (500µl / 1L)

Ponceau S: 0.1% w/v in 1% Acetic acid, reusable

Transfer buffer or CAPS buffer Stripping buffer

25mM Tris 5X CAPS 2%SDS

192mM glycine 11.1g / L 100mM ßME

0.1% SDS pH to 11 w/ NaOH 50mM Tris-HCl

20% MeOH 1X CAPs pH 7.0

(no need to pH 200ml MeOH

8.3) 200ml 5X + 600ml ddH2O

Procedure:

Transfer proteins from gel to membrane (nitrocellulose or PVDF):

1. Pour some transfer buffer (or CAPS buffer) into a container so that half of the cassette and sponges are 0.5cm below the surface of the buffer.

2. After gel finishes running, take off the white plate, cut off the wells, lay nitrocellulose over the gel and 2 pieces of filter paper on top. Now peel off the gel from the glass plate. Clean the debris by lifting one side of the gel at a time, folding it over and remove all the bubbles. Lay the other pieces of filter paper above the gel. Put into the cassette with the gel closer to the black(-) plate and membrane to red(+) plate. Insert into the chamber.

3. Pour transfer buffer (or CAPS buffer) into the chamber up to the edge. Bring to the cold room (in ice) along with the power supply. The buffer can't get heated up, otherwise the gel will stick to nitrocellulose.

4. If transfer o/n, turn the power to 20V 40mA. Otherwise turn up to 70V106mA and let run for ~3hrs. Set the voltage first and then convert to mA to check if the circuit is complete. To check if the proteins are all transferred to the nitrocellulose, lift one corner of the gel to see if any blue is left in the gel. In other words, the gel should be clear when it's done transferring.

(Optional) Stain blot for proteins using ponceau S. Wash blot briefly in water then incubate in ponceau S solution for 1 min. Drain off solution and rinse in water until background is reduced. Photocopy blot for a permanent record (Lay the gel directly onto the copier and a piece of transparency over it. When finished, wipe off the copier). Rinse blot several times with TBS-Tween to remove ponceau S.

Block blot in 5% milk/TBS/Tween in the container, 2hr, RT, on shaker; OR o/n in 4oC on a rocker.

Primary antibody probing: dilute antibody in 5% milk/TBS, incubate with blot, 2hr RT, or o/n in cold room on a rocker. Cut open 3 sides of a sealable bag, lay flat on bench, lay the gel, seal 3 sides, fill in antibody sol'n (6ml), roll out the bubbles with a pipette, seal 4th side finally. Tape the gel bag onto the rocker.

Wash: 3 x 5' TBS/Tween

Secondary antibody: dilute antibody in 5% milk/TBS, incubate 2hr, RT, see primary

Wash: 3 x 10' TBS/Tween, and 1 x 5' in TBS

Detection / Chemiluminescence: cut a pc. of transparency into 2 smaller pcs. that fit into the cassette; mix oxidizing and luminol reagent (from Dupont NEN) 1: 1, transfer onto a piece of parafilm, lay the blot over the sol'n 1min; then insert the blot btwn the 2 transparencies. NO BUBBLES!

Expose: initially try for 1min and then adjust for the later trial. The signals DO decay over time.

P.S. If still no presumed bands visible, then rinse and store in PBS/Tween o/n and repeat the blocking and probing.

Stripping and reprobing Western blots:

Note: This protocol works well with chemiluminescence and radioactive detection, but is not expected to be useful with colorimetric detection because of the production of an insoluble reaction product in that method of detection.

Rinse the blot briefly in water.

Wash 2 X 10' in TBS/Tween

Reblock 1hr, RT in 5% milk/TBS/Tween

Stripping: add ~12ml stripping buffer to the blot in a bag, incubate in 50oC water bath for 30' with occasional agitation

Wash blot 2 x 30" + 2 x 10' in TBS/Tween

Incubate (optional) the blot with detection reagent and expose to film to ensure that the blot has been adequately stipped (can also redo incubation in 2o before detection to make sure all 1o Ab has been removed).

Block for 2 hr, RT in 5% milk/TBS/Tween as usual then go into 1o, etc.