Protocol for Sandwich ELISA for the Detection of Membrane Pr

Materials

• Maxisorb 96-well plate
• Goat anti-mouse IgG, unconjugated
• Goat anti-rabbit IgG, peroxidase conjugated
• Specific monoclonal mouse antibody as capture antibody
• Specific polyclonal rabbit antibody as detector antibody
• Protease inhibitors (suggested: 1 mM PMSF, 1 µg/ml Aprotinin, 1.5 µM Pepstatin A)
• Microplate absorbance reader with filters at 450 nm and a reference wavelength (e.g. 620 - 650 nm)

Solutions

• Coating buffer: 0.1 M Na-carbonate, pH 9.6 (store 0.5 M stock at -20°C)
• Blocking buffer A: 1% tryptone/peptone from casein (TP) in 0.1 M Na-carbonate, pH 9.6
• Washing buffer: Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST)
• Solubilization buffer A: 10% sodium dodecyl sulfate (SDS) in PBS
• Solubilization buffer B: 1.2% Triton X-100 in PBS
• Antigen buffer A: 0.2% Triton X-100/0.05% TP in TBS
• Antigen buffer B: 0.05% TP in TBS
• Blocking buffer B: 0.5% TP/0.5% BSA/0.5% gelatin in TBST
• Substrate solution: Tetramethylbenzidine (TMB) reagent for development
• Stop solution: 0.25 M H2SO4 to stop development

Procedure

1. Coat 96-well microplate with 100 µl goat anti-mouse IgG (1 µg/ml) in coating buffer and incubate for 3 h at RT and 700 rpm.
2. Block the surface with blocking buffer A for 1 h at RT and 700 rpm.
3. Wash the plate three times with washing buffer (at least 5 min per wash) and transfer them to 4°C.
4. Apply monoclonal capture antibody diluted in washing buffer and incubate overnight at 4°C. Dilute ascites 1 : 4000, purified antibody 1 : 2000 (50 - 75 ng/well).
5. Antigen solubilization: Adjust protein standard and samples to 3 mg/ml total protein and a final SDS concentration of 1.2% with solubilization buffer A and rotate 15 min at RT. Add 5 volumes of ice-cold solubilization buffer B to each sample and rotate 15 min at 4°C. Pellet the insoluble fraction at 100,000 x g for 30 min (acceptable alternative: 13,000 rpm for 30 min at 4°C in a tabletop centrifuge) and transfer the supernatant to a new tube. Dilute the supernatant in antigen buffer B to 0.2% Triton concentration.
   Note: If complete tissue samples are used, DNase should be added as 0.1 µg/µl together with protease inhibitors, and SDS should be added as last component after mixing everything else.
6. Wash the plate once with washing buffer, twice with antigen buffer A at RT.
7. Apply antigen diluted in antigen buffer A and incubate for 2 h at RT and 700 rpm.
8. Wash twice with antigen buffer A, once with blocking buffer B.
9. Incubate with blocking buffer B for 30 min at RT.
10. Apply polyclonal detector antibody diluted in blocking buffer B (dilution 1 : 1000 up to 1 : 8000) and incubate for 1 h at RT and 700 rpm.
11. Wash three times with blocking buffer B.
12. Incubate with HRP-coupled goat anti-rabbit antibody, diluted 1 : 10,000 in blocking buffer B, for 1 h at RT and 700 rpm.
13. Wash three times with washing buffer.
14. Add 100 µl substrate buffer for development.
15. Stop the reaction after 30 min with 100 µl stop solution and read the absorbance at 450 nm (ref: 620 - 650 nm).

Reference

Geumann C, Grønborg M, Hellwig M, Martens H & Jahn R (2010). A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins. Analytical Biochemistry 402: 161-9.