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  4. Western Blotting Protocol

Western Blotting Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 mL 20X PBS to 950 mL dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 mL 10X to 900 mL dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723); Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 mL 10X running buffer to 900 mL dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X transfer buffer: add 100 mL 10X transfer buffer to 200 mL methanol + 700 mL dH2O, mix.
  6. 10X Tris Buffered Saline with Tween 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 mL 10X TBST to 900 mL dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 mL, add 7.5 g nonfat dry milk to 150 mL 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody product webpage; for 20 mL, add 1.0 g BSA or nonfat dry milk to 20 mL 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Generally, a pore size of 0.2 µm is recommended.
  15. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076).
  16. Detection Reagent: LumiGLO chemiluminescent reagent and peroxide (#7003) or SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µL per well of 6-well plate or 500 µL for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µL sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µL onto SDS-PAGE gel (10 cm x 10 cm).
    NOTE: It is recommended that prestained molecular weight markers (#59329, 5 µL/lane) be loaded to verify electrotransfer and a biotinylated protein ladder (#7727, 10 µL/lane) be loaded to determine molecular weights.
  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; adjust volumes accordingly for different-sized membranes.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 mL TBS for 5 min at room temperature.
  2. Incubate membrane in 25 mL of blocking buffer with gentle agitation for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 mL of TBST.

II. Primary Antibody Incubation

Proceed to one of the following specific set of steps depending on the primary antibody used.

For Unconjugated Primary Antibodies

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 mL primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 mL of TBST.
  3. Incubate membrane with the species-appropriate HRP-conjugated secondary antibody (#7074 or #7076 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 mL of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 mL of TBST.
  5. Proceed with detection (Section D).

For HRP-Conjugated Primary Antibodies

  1. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 mL primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 mL of TBST.
  3. (Optional) To detect biotinylated protein markers, incubate with Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) in 10 mL of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 mL of TBST.
  5. Proceed with detection (Section D).

For Biotinylated Primary Antibodies

  1. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 mL primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 mL of TBST.
  3. Incubate membrane with Streptavidin-HRP (#3999 at the appropriate dilution) in 10 mL of blocking buffer with gentle agitation for 1 hr at room temperature. 
    NOTE: Streptavidin-HRP visualizes biotinylated protein markers. Additional incubation with Anti-Biotin, HRP-Linked Antibody is unnecessary.
  4. Wash three times for 5 min each with 15 mL of TBST.
  5. Proceed with detection (Section D).

D. Protein Detection

  1. Incubate membrane with 10 mL LumiGLO (0.5 mL 20X LumiGLO #7003, 0.5 mL 20X peroxide, and 9.0 mL purified water) or 10 mL SignalFire™ #6883 (5 mL Reagent A and 5 mL Reagent B) with gentle agitation for 1 min at room temperature.
  2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap, and expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time.
    NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr.