BioProtocols--western1

Protocol 1: Western Blot 1

1) Sample protein preparation

2) Protein concentration assay

3) Electrophoresis and blotting

4)Blocking non-specific antigen

5) Incubation with primary antibody

6) Incubation with secondary antibody

7) Substrate (ECL) incubation

Restore a western blot using pierce stripping buffer

Western blot troubleshooting

Protocol 2: Western Blot 2.

Protocol 3: Western Blot 3.

More online Western blot protocols

Western Blot Protocol

Sample protein preparation

Monolayer Cells

Grow cells to 80-90% confluency in a 100 mm dish, remove culture medium and rinse cell monolayer with 1x PBS at room temperature.

Add 0.3-0.5 ml lysis buffer enriched with protease inhibitor cocktail to the monolayer cells in the plate. Scrape the cells off and transfer the lysate to an eppendorf tube. Incubate for 30 minutes on ice.

Centrifuge cell lysate at 14000 rpm for 15 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new eppendorf tube. This is your whole cell lysate.

Tissue Samples

Weigh tissue and cut into very small pieces. Frozen tissue should be sliced very thinly and thawed in lysis buffer containing proteinase inhibitors.

Sonicate the tissue, and incubate on ice for 30 minutes. Transfer to eppendorf tubes, centrifuge at 14000 rpm for 15 minutes at 4° C. The supernatant fluid is the total cell lysate.

Make protein concentration assay for each sample.

Electrophoresis and blotting
a. Add an appropriate amount of loading buffer to each sample. Vertex the tubes.
b. Boil at water bath for 5 minutes.
c. Load 5-100 ug total protein in a volume that is appropriate for the size of the wells. Load 10 ul protein marker to 1-2 blank lanes
d. Electrophorese proteins for the appropriate time and voltage according to the manufacturer’s instruction.
e. Transfer proteins from the gel to a suitable membrane (e.g. nitrocellulose, PVDF, etc.) following standard method.
Blocking non-specific antigen
a. Remove the membrane from the transfer apparatus and rinse in TBST to remove loose acrylamide.
b. Transferred proteins can be visualized by staining the membrane for 3-5 minutes with Ponceau S.
c. Remove the dye from the membrane by washing with TBST.
d. Place the membrane into 5% non-fat dry milk blocking solution.
e. Block for 1 hour at room temperature, or overnight at 4 ° C.
Incubation with primary antibody
a.Discard blocking buffer and add the antibody, diluted in blocking buffer at a proper ratio.
b. Incubate with agitation for 1 hour at room temperature or overnight at 4°C.
Incubation with secondary antibody
a. Wash for 20 minutes with agitation in wash buffer (TBS with 0.1% Tween 20), changing the wash buffer every 5 minutes.
b. Discard the wash solution and add HRP-conjugated secondary antibody, diluted in 5% non-fat dry milk wash buffer.
c. Incubate for 1 hour at room temperature.
d. Discard the antibody conjugate and wash for 40 minutes with agitation in wash buffer (TBS with 0.1% Tween 20), changing the wash buffer every 10 minutes.
Substrate (ECL) incubation
a. Discard washing buffer and add the freshly made ECL mixture for 1-5 minutes. Make sure the membrane is covered with the ECL mixture.
c. Remove the blot from the tray and place it between two pieces of write-on transparency film.
d. Smooth over the covered blot to remove air bubbles and excess substrate.
e. Expose to X-ray film.