Protocol for sandwich ELISA for the detection of membrane proteins

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
Tetramethylbenzidine (TMB) reagent for development
Microplate absorbance reader with filters at 450 nm and a reference wavelength (e.g. 620-650 nm)

Solutions:

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

Procedure:

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).

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.