18 Western blot protocol

Western blot protocol

1. Run your protein samples on a 12% polyacrylamide gel as previously performed.
   • Use 5 uL of sample. Don’t forget to include your protein ladder (5 uL).
   • Make notes in your lab notebook on your voltage and any other key volumes.

2. Transfer your gel to a membrane and block the membrane

• For each gel you are transferring, you need 1 nitrocellulose membrane, 2 pieces of filter paper, and 2 sponges.
• Prepare transfer buffer: Buffer should contain 1X Tris-Glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3), with 20% methanol. For the Biorad transfer system, you need ~1.2 L of transfer buffer. Chill before use.

You already prepared your 10X Tris-Glcyine buffer, so use the table below to determine how much of each solution to combine:

• Equilibrate nitrocellulose membrane in transfer buffer for ~ 5 min. Handle the membrane with clean forceps only—do not use your fingers.
• Add ~1 cm depth of blotting buffer to a plastic container and place plastic cassette inside with black side down.
• Set up sandwich on top of cassette in the following order in the dish of transfer buffer, rolling out air bubbles at each step, and pre-wetting all components.
  • Fiber sponge
  • filter paper
  • gel (remove from plastic casing with spatula, rinse in water, be careful of orientation of gel—ladder will be on right side of gel)
  • membrane
  • filter paper
  • fiber sponge

• Close the cassette with the white clip and place in gel box. Make sure the black side of the cassette faces the black side of the electrode module.
• Fill up box to Blotting line with Transfer buffer

• Transfer at 100V for 30 minutes.
• While transfer is running, prepare PBST solution and non-fat milk solution
• PBST= 1X phosphate buffered saline (PBS) with 0.025% Tween 20 (a detergent). This is your Western blot WASH BUFFER that washes away unbound antibodies and helps ensure your antibodies only stick to their target protein.
• After transfer is complete, take apart sandwich and put membrane into clean plastic box
• Blocking: Block in 5% non-fat milk dissolved in PBST in for >30 min at RT. If longer than 2-3 hrs, perform blocking step at 4° C.

3. Probing and developing your western blot

Note: Do all washes on rocker.

• Prepare primary antibody by adding 50 uL of antibody (200X) to 10 mL of 5% non-fat milk (in PBST) in a 15 mL tube.
• Immerse membrane in 10 mL of primary antibody for 20 min
• Save the primary antibody in the 15 mL tube in case something goes wrong. Rinse membrane with wash buffer (fast wash)
• Do three 5 minute washes (slow washes)
• Incubate blot with 10 mL of 5% non-fat milk containing 50 uL of secondary antibody for 15 minutes.
• Do one fast wash with wash buffer
• Do three 5 minute washes with wash buffer.
• Discard the wash and add 10 mL of HRP color detection reagent
• Incubate for ~15 minutes, and watch color development
• Rinse the membrane twice with di-water and blot dry with paper towel
• Air dry and then cover in plastic wrap and tape into your lab book.