Protocol for sandwich ELISA for the detection of membrane proteins
- 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
- Tetramethylbenzidine (TMB) reagent for development
- Microplate absorbance reader with filters at 450 nm and a reference wavelength (e.g. 620-650 nm)
- 0.1 M Na-carbonate, pH 9.6 (store 0.5 M stock at -20°C)
- 1 % tryptone/peptone from casein (TP) in 0.1 M Na-carbonate, pH 9.6
- Tris-buffered saline (TBS) with 0.05 % Tween 20 (TBST)
- Phosphate-buffered saline (PBS), 1.2 % Triton X-100 in PBS, protease inhibitors, and 10 % sodium dodecyl sulfate (SDS) for antigen solubilization
- 0.2 % Triton X-100/0.05 % TP in TBS (antigen buffer)
- 0.05 % TP in TBS (antigen buffer w/o Triton)
- 0.5 % TP/0.5 % BSA/0.5 % gelatin in TBST (blocking buffer)
- 1 M H2SO4 to stop development
- Coat 96-well microplate with 100 µl goat anti-mouse IgG (1 µg/ml) in 0.1 M Na-carbonate, pH 9.6, and incubate 3 h at RT and 700 rpm.
- Block the surface with TP in carbonate buffer; 1 h at RT and 700 rpm.
- Wash plates three times with TBST (at least 5 min per wash) and transfer them to 4°C.
- Apply monoclonal capture antibody diluted in TBST and incubate over night at 4°C. Dilute ascites 1 : 4000, purified antibody 1 : 2000 (50-75 ng/well).
- Antigen solubilization: Adjust protein standard and samples to 3 mg/ml total protein in PBS containing 1.2 % SDS and protease inhibitors (suggested: 1 mM PMSF, 1 µg/ml Aprotinin, 1,5 µM Pepstatin A) and rotate 15 min at RT. Add 5 volumes of ice-cold 1.2 % Triton X-100 in PBS to each sample and rotate 15 min at 4°C. Pellet the insoluble fraction at 100,000g 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 (without and with Triton X-100 to adjust to 0.2 % Triton).
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.
- Wash the plate once with TBST, twice with antigen buffer at RT.
- Apply antigen diluted in antigen buffer; 2 h at RT and 700 rpm.
- Wash twice with antigen buffer, once with blocking buffer.
- Incubate with blocking buffer for 30 min at RT.
- Apply polyclonal detector antibody diluted in blocking buffer (dilution: 1 : 1000 up to 1 : 8000); 1 h at RT and 700 rpm.
- Wash three times with blocking buffer.
- Incubate with HRP-coupled goat anti-rabbit antibody, diluted 1 : 10000 in blocking buffer, for 1 h at RT and 700 rpm.
- Wash three times with TBST.
- Add 100 µl TMB reagent for development.
- Stop the reaction after 30 min with 100 µl 1 M H2SO4 and read the absorbance at 450 nm (ref: 620-650 nm).
, 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.