PathScan^® Sandwich ELISA Antibody Pair Protocol

A. Required Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. Coating Buffer: 1X PBS, (20X PBS #9808)
   3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, pH 7.4.
2. Wash Buffer: 1X PBS/0.05% Tween-20, (20X PBST #9809).
3. Blocking Buffer: 1X PBS/0.05% Tween-20, 1% BSA.
4. 1X Cell Lysis Buffer: (10X Cell Lysis Buffer #9803)
   This buffer can be stored at 4°C for short-term use (1–2 weeks).
   Recommended: Add 1mM phenylmethylsulfonyl fluoride (PMSF) immediately before use.
   • 20 mM Tris (pH 7.5)
   • 150 mM NaCl
   • 1 mM ethylene diamine tetraacetate (EDTA)
   • 1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA)
   • 1% Triton X-100
   • 2.5 mM sodium pyrophosphate
   • 1 mM β-glycerophosphate
   • 1 mM Na₃VO₄
   • 1 µg/ml leupeptin
5. TMB Substrate: (#7004)
6. STOP Solution: (#7002)

NOTE: Reagents should be made fresh daily.

B. Coating Procedure

1. Rinse microplate with dH₂O. Add 200 µl of dH₂O and discard liquid. Blot on paper towel to make sure wells are dry.
2. Dilute capture antibody 1:100 in PBS. For a single 96 well plate, add 100 µl of Capture Antibody Stock to 9.9 ml PBS. Mix well and add 100 µl/well. Cover plate and incubate overnight at 4°C (17–20 hours).
3. After overnight coating, gently uncover plate and wash wells:
   1. Discard plate contents into a receptacle.
   2. Wash 4 times with Wash Buffer, 200 µl each time for each well. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
   3. Clean the underside of all wells with a lint-free tissue.
4. Block plates. Add 150 µl of Blocking Buffer/well, cover plate, and incubate at 37°C for 2 hours.
5. After blocking, wash plate as in Step 3. Plate is ready to use.

C. Preparing Cell Lysates

1. Aspirate media, treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm in diameter) and incubate the plate on ice for 5 minutes.
4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
5. Sonicate lysates on ice.
6. Microcentrifuge for 10 minutes at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at –80°C in single-use aliquots.

D. Test Procedure

1. Lysates can be used undiluted or diluted in Blocking Buffer. 100 µl of lysate is added per well. Cover plate and incubate at 37°C for 2 hours.
2. Wash plate as in Coating Procedure, Step 3.
3. Dilute detection antibody 1:100 in Blocking Buffer. For a single 96 well plate, add 100 µl of Detector Antibody Stock to 9.9 ml of Blocking Buffer. Mix well and add 100 µl/well. Cover plate and incubate at 37°C for 1 hour.
4. Plate is washed as in Coating Procedure, Step 3.
5. Secondary antibody, either anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in Blocking Buffer. For a single 96 well plate, add 10 µl of secondary antibody stock to 9.99 ml of Blocking Buffer. Mix well and add 100 µl/well. Cover and incubate at 37°C for 30 minutes.
6. Wash plate as in Coating Procedure, Step 3.
7. Add 100 µl of TMB Substrate per well. Cover and incubate at 37°C for 10 minutes.
8. Add 100 µl of STOP Solution per well.
9. Read plate on a microplate reader at Absorbance 450 nm.