Yu:Western

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Western Blotting

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Contents


SDS PAGE

1.Cast SDS-PAGE gel and assemble the gel apparatus.
2. Add 10μL of desired sample and 5μL 3x Gel loading buffer to an eppendorf tube and boil for 5 minutes. Do this in separate tubes for each sample to be analyzed. After boiling, samples may be kept at room temperature until loaded
3. Load 12μL to 15μL of sample into each lane. Load protein prestain marker, as well as BenchMark, if desired. Load any empty lanes with 1x Gel loading buffer
4. Run gel until the loading buffer is just running off the bottom of the gel

Transfer

1.Make 1L 0.5x TRIS-Glycine Western Transfer Buffer from 10x stock and assemble the western transfer tank.
2. Carefully assemble the transfer sandwich, rolling to remove air bubbles at each step.
Black (Negative) Side
  1. Sponge
  2. 2 pieces of Whatman chromatography paper
  3. SDS-PAGE Gel
  4. Nitrocellulose membrane
  5. 2 pieces of Whatman chromatography paper
  6. Sponge
Red (Positive) Side
3. Transfer at room temperature for 1 hour at constant 100 volts. Current should range from 200-350 mA.
4. Remove nitrocellulose membrane and Ponceau stain in a Western blotting tray. Mark any protein ladders with a dull #2 pencil.

Immunoblotting

1. Make blocking buffer for Western blot and stir until fully dissolved. Store at 4°C
Solution Quantity
Carnation instant milk 5g
PBST 100mL
2. Block nitrocellulose membrane on rocker for 20-30 minutes at room temperature with blocking buffer, or overnight at 4°C.
3. Discard blocking buffer and add primary antibody. Incubate on rocker at room temperature for 1 hour, or overnight at 4°C.
4. Draw off and save primary antibody for reuse. Rinse with 30-50mL PBST.
5. Wash twice on rocker for 10 minutes in PBST.
6. Make secondary antibody (2μL into 10mL Western Blocking Buffer) and incubate membrane on rocker at room temperature for 1 hour.
7. Discard secondary antibody. Rinse with 30-50mL PBST
8. Wash twice on rocker for 10 minutes in PBST.
9. Wash with distilled H2O for 10 minutes.
10. Develop and image.

Stripping

Stripping Buffer

Solution Volume
1.5M Tris pH 6.8 2.08 mL
β-Mercaptoethanol 0.5 mL
10% SDS 10 mL
milliQ H2O 37.42 mL
Final Volume is 50 mL

Procedure

  1. Wash Membrane for 15-20 min in 1x PBST at room temperature to wash away developing solution
  2. Incubate membrane in 50 mL of stripping buffer on a shaking platform for 30 min at 50°C
  3. Wash Membrane for 15-20 min in 1x PBST at room temperature
  4. Block and reprobe membrane with desired antibody
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