Western Blot (WB) Protocol

Instructions

Western blot (WB) is a technique used to identify and localize proteins based on their binding ability to specific antibodies. First, proteins are denatured and then separated by SDS-PAGE gel electrophoresis. The separated proteins are then stained by the PVDF or nitrocellulose membrane, and then blocked with milk (or another blocking reagent). The membrane is then stained with a secondary antibody specific to the primary antibody, which can be detected by a variety of methods. When compared to a ladder or size marker (in kDA), protein size and expression can be determined.

Solution and reagents

For the protein weight lower than 20 kD, Trincine buffer is recommended, the recipe as follow:

** If the weight of target protein is bigger than 100 kD, we recommend adding 0.04% SDS in transfer buffer, and to reduce the methanol percentage to 10% or lower.

Sample preparation

Preparation for lysate of cell culture

1. Place cell culture dish on ice and wash cells with ice-cold sterile PBS.
2. Lyse cells by adding RIPA buffer (1 mL for per 10cm dish) containing fresh protease inhibitors. Keep on ice for 15-30 min. Scrape cells and transfer to a microfuge tube.
3. Sonicate the cell suspension to shear DNA and complete cell lysis (the time of sonication varies with cell type).
4. Spin at 12,000 RPM for 15-20 min at 4℃.
5. Transfer the supernatant into a new tube. Keep on ice at all times.
6. Use BCA assay to measure the protein content in the supernatant.
7. Add 6x sample loading buffer to the supernatant depending on the protein content and amount of protein needed to load on.
8. Boil samples for 5 min at 95℃ (unless noted otherwise).
9. Centrifuge for 5 min 12,000 RPM and store at -20℃.

Preparation for lysate of tissue

1. Dissect the required tissues on ice and wash away the excess blood.
2. Place the fresh tissue in a clean mortar, use liquid nitrogen to grind tissue. Collect the tissue powder into a precooling tube.
3. Add RIPA buffer according to the amount of tissue (500μl for 50 mg tissue) and incubate on ice for 15-30 min with intermittent mixing.
4. Sonicate the supernatant on ice (time varies depending on the tissue), then incubate on ice for 15-20 min.
5. Spin at 12,000 rpm for 15-20 min at 4℃.
6. Transfer the supernatant into a new tube. Keep on ice at all times.
7. Use BCA assay to measure the protein content in the supernatant.
8. Add 6x sample loading buffer to the supernatant depending on the protein content and the amount of protein needed to load on.
9. Boil samples for 5 min at 95℃ (unless noted otherwise).
10. Centrifuge for 5 min 12,000 RPM and store at -20℃.

NOTE: To avoid protein degradation, perform the entire process on ice.  Make sure that the protein of interest is present in your samples.

Preparation of Gel

1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (~20 ml). Ensure there are no bubbles.
4. Seal with 1 ml water-saturated 1-butanol.
5. When gel has set, pour off the butanol and rinse with deionized water.
6. Pour the stacking gel (~5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Flush the wells out thoroughly with running buffer before running the gel.
9. Follow the table below to determine which gel content you require.

Running the gel

1. Flash-spin the samples to remove impurities.
2. Load 10-30 μg of total protein into the wells. We recommend 10-100 ng of purified proteins. Loading quantities should be adjusted according to the result.
3. Molecular weight markers should be included in a lane to indicate the protein of interest.
4. Run with constant voltage (voltage set at 120 V or lower). To obtain a better experimental result, 80v is recommended when the whole protein migrates in concentration gel. While the proteins migrate into separation gel, voltage should be boosted up.
5. Usual running time is about 80 mins, however, if the protein weight is lower then 20 kD, the runtime should be shorter, and if the protein weight is larger than100 kD, the runtime should be longer to ensure a better separation of proteins.

Transfer and Blocking

1. Carefully separate the gel.
2. Layer filter paper-gel- filter paper, creating a “sandwich” (see image below). Keep the “sandwich” hydrated and avoid bubbling between layers:
   


Western Blot layers courtesy of Cusabio. Click to enlarge.

3. Assemble the transfer device, and transfer proteins to nitrocellulose or PVDF membrane. We recommend a 0.22 μm membrane for proteins with weights lower than 20 kD.
4. There are two transfer methods: wet transfer or semi-dry-transfer. Both methods work well for high molecular weight and low molecular weight proteins. However, semi-dry-transfer can yield a higher background staining.
5. Rinse the blot in PBS for approximately 5 mins.

Block the membrane using 1% casein in PBS (may use 1% BSA or 5% non-fat dry milk) for 1 hour at room temperature.

Antibody incubation

1. Dilute the primary antibody in blocking buffer and incubate 1-3 hours at room temperature, or overnight at 4℃. We recommend incubating at a low temperature over a long period of time, as overnight incubation typically yields improved antibody binding.
2. Wash the membrane in PBST for 10 min for three times and apply the diluted conjugated secondary antibody in blocking buffer (as per manufacturer’s instructions) and incubate 1 hour at room temperature.
3. Wash the blot in PBST three times for 10 min each.

Detection

1. Apply the detection reagent of choice in accordance with the manufacturer’s instructions.
2. To minimalize signal reduction, mix chemiluminescent reagents in prior to use to limit light exposure and degradation.