Materials

• Membrane: Nitrocellulose (0.45mm), polyvinylidene difluoride (PVDF), or nylon.
• Whatman 3MM filter papers, cut to size of gel for transferring.
• Transfer buffer, pH 8.3 (Molecular Cloning, 1989, p.18.65 by Maniatis et al.)
  • 39 mM Glycine
  • 48 mM Tris base
  • 0.037% SDS (electorophoresis grade)
  • 20% Methanol
• PBS, pH 7.5
• PBST, pH 7.5: PBS containing 0.1% Tween-20
• Blocking reagent:
  • 2% BSA in PBS or 2-5% Carnation fat-free dry milk with 0.17% NaN₃
• Ponseau S
  • Stock solution (10X): 2 gm Ponceau S; 30 gm Trichloroacetic acid; 30 gm
  • Sulfosalicylic acid; water to 100 ml
• Primary antibody prepared at working dilution in PBST
• Conjugated secondary antibody at working dilution in PBST
• Chromogenic or chemiluminescence visualization systems
• Stripping Buffer
  • 100 mM mercaptoethonal
  • 2% SDS
  • 62.5 mM Tris HCL, pH 6.7

Setting up the transfer sandwich

1. At the end of the electrophoresis, trim polyacrylamide from top wells of gel (optional), then float gel into transfer buffer and shake for 10 min.
2. Soak the Whatman paper, mesh screens and nitrocellulose or PVDF membrane in transfer buffer for 10 min.
3. Assemble transfer apparatus according to manufacturer's instructions. Assemble the parts in the following order from cathode to anode:
   • plastic grids
   • cathode
   • plastic grid
   • mesh screen
   • wet three filters
   • Gel
   • wet nitrocellulose membrane
   • wet three filters
   • mesh screen
   • plastic grid
   • anode
   • solid plastic piece
   
   Carefully insert the entire assembly into the transfer box and make sure that order is correct: cathode - gel - membrane- anode. Attach red electrode to anode and black to cathode.
   
   Time and voltage of transfer are dependent on apparatus in use. High molecular weight proteins ( more than 80-100 kDa) transfer slowly (usually overnight, 15-20V), medium and low molecular weight proteins transfer faster ( 2-6 hrs, 20-25V).
4. After transfer rinse membrane in PBS.
5. Ponceau S staining can be done to reveal a protein profile on membrane. Stain membrane 10 min with 1X Ponceau S (diluted in water), discard Ponceau S solution, rinse membrane several times with large volume PBS or water (100-150 ml) to remove red background. Visualize by copying on Xerox machine and remove Ponceau S staining completely by washing in PBS or in water.( see Molecular Cloning, 1989, p.18.67 by Maniatis at al.)
6. Block membrane in blocking reagent overnight at +4^oC or 2 hrs at room temperature.

Developing membrane with antibody

1. Rinse membrane with PBST twice and incubate in primary antibody 1-2 hrs at RT or overnight at +4^oC. Note that a flat rotating shaker may give better coverage of the membrane for small volumes of antibody solution.
2. Wash in large volume PBST (50-100ml) 10 min, 3 times.
3. Incubate with secondary conjugated antibody 1-2 hrs at RT.
4. Wash in large volume PBST (50-100ml) 10 min, 3 times.
5. Secondary antibodies are conjugated (labeled) with biotin or enzymes which are horseradish peroxidase (HRP) and with alkaline phosphatase (AP). Choose appropriate chromogenic or luminescent substrate systems for visualization (see Current Protocols in Protein Science,1998, Chapter 10.10.5 ).

Visualization with Cromogenic substrates

1. After incubation with primary and secondary antibodies, the membrane is placed in the appropriate substrate solution and protein bands appear a few minutes.
2. Terminate reaction by washing membrane in distilled water, air dry and photograph for permanent record.

Visualization with luminescent substrates

1. Place blot in a substrate solution containing luminol for HRP system, or dioxetane phosphate for AP (alkaline phosphatase) system (see Current Protocols in Protein Science,1998, Chapter 10.10.6).
2. Cover membrane with plastic wrap and exposure to film from few minutes to several hours. Develop film.

Reprobing Blot

Membrane may be stripped and reprobed with a different antibody.
Submerge the moist membrane in stripping buffer, incubate 2 x 15 min in 100ml stripping buffer at 55^oC.
Rinse 3 x 15 min at room temperature with 100 PBST.
Block membrane 1 hr at room temperature with BSA or skim milk and re-probe with alternative antibody as described above.

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