Western Blot Standard Protocol

Solutions and Buffers

Lysis Buffers

These buffers can be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.

1. NP-40 Buffer

   - 150 mM NaCl

   - 1.0% NP-40 (can be replaced with 0.1% Triton X-100)

   - 50 mM Tris-HCl, pH 8.0

   - Protease inhibitors

2. RIPA Buffer

   - 150 mM NaCl

   - 1% IGEPAL CA-630

   - 0.5% sodium deoxycholate

   - 0.1% SDS (sodium dodecyl sulphate)

   - 50 mM Tris-HCl, pH 8.0

   - Protease inhibitors

3. Tris-HCl Buffer

   - 20 mM Tris-HCl

   - Protease inhibitors

Running, Transfer, and Blocking Buffers

1. Laemmli 2X Buffer/Loading Buffer

   - 4% SDS

   - 10% 2-mercaptoethanol

   - 20% glycerol

   - 0.004% bromophenol blue

   - 0.125 M Tris-HCl

   - Adjust pH to 6.8

2. Running Buffer (Tris-Glycine/SDS)

   - 25 mM Tris base

   - 190 mM glycine

   - 0.1% SDS

   - Adjust pH to 8.3

3. Transfer Buffer (Wet)

   - 25 mM Tris base

   - 190 mM glycine

   - 20% methanol

   - Adjust pH to 8.3

   - For proteins larger than 80 kDa, add SDS to a final concentration of 0.1%

4. Transfer Buffer (Semi-dry)

   - 48 mM Tris

   - 39 mM glycine

   - 20% methanol

   - 0.04% SDS

5. Blocking Buffer

   - 3–5% milk or BSA (bovine serum albumin) in TBST buffer

   - Mix well and filter to avoid spotting during color development

Sample Preparation

Cell Culture Lysate

1. Place the cell culture dish on ice and wash the cells with cold PBS.

2. Remove the PBS and add cold lysis buffer (1 mL per 107 cells/100 mm dish/150 cm2 flask; 0.5 mL per 5x106 cells/60 mm dish/75 cm2 flask).

3. Scrape cells off the dish using a cold plastic cell scraper and transfer the suspension to a pre-cooled microcentrifuge tube. Alternatively, trypsinize cells, wash with PBS, and resuspend in lysis buffer.

4. Agitate constantly for 30 min at 4°C.

5. Centrifuge the suspension at 4°C. Adjust centrifugation force and time based on cell type (e.g., 5 min at 14,000–17,000 g for most cells; light centrifugation for leukocytes).

6. Carefully remove the tubes from the centrifuge, place on ice, and transfer the supernatant to a fresh tube on ice. Discard the pellet.

Tissue Lysate

1. Dissect the tissue of interest quickly on ice using clean tools to prevent protease degradation.

2. Place the tissue in round-bottom microcentrifuge or Eppendorf tubes and snap freeze in liquid nitrogen. Store at -80°C for later use or keep on ice for immediate homogenization.

3. For a ~5 mg tissue piece, add ~300 μL of cold lysis buffer, homogenize with an electric homogenizer, and rinse the blade twice with 2 x 200 μL lysis buffer. Agitate constantly for 2 h at 4°C.

4. Centrifuge for 5–10 min at 14,000–17,000 g at 4°C. Carefully remove the tubes, place on ice, and transfer the supernatant to a fresh tube on ice. Discard the pellet.

Sample Preparation

1. Remove a small volume of lysate for protein quantification and determine the protein concentration for each lysate.

2. Calculate the amount of protein to load and add an equal volume of 2X Laemmli sample buffer.

3. To reduce and denature samples, boil each lysate in sample buffer at 100°C for 5 min. Aliquot and store at -20°C for future use if needed.

Gel Electrophoresis and Transfer

1. Load equal amounts of protein (20–30 μg of total protein from cell lysate or tissue homogenate, or 10–100 ng of purified protein) into the wells of an SDS-PAGE gel, along with a molecular weight marker.

2. Run the gel for 1–2 h at 100 V. Optimize time and voltage as needed and follow the manufacturer's instructions. Use a reducing gel unless the antibody datasheet recommends non-reducing conditions.

3. Choose the appropriate gel percentage based on the size of your protein of interest (see table in the original protocol).

4. Transfer the protein from the gel to a nitrocellulose or PVDF membrane. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack.

5. Prepare the transfer stack as shown in the example image (see original protocol).

6. Optimize transfer time and voltage following the manufacturer's instructions. Check protein transfer using Ponceau S staining before blocking.

Antibody Staining

1. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking buffer.

2. Incubate the membrane with appropriately diluted primary antibody in blocking buffer. Overnight incubation at 4°C is recommended, but other conditions can be optimized.

3. Wash the membrane three times with TBST for 5 min each.

4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.

5. Wash the membrane three times with TBST for 5 min each.

6. Develop the signal according to the kit manufacturer's recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.

7. Acquire the image using darkroom development techniques for chemiluminescence or standard image scanning methods for colorimetric detection.