PathScan^® Sandwich ELISA Protocol

A. Reagent Preparation

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

1. Bring all microwell strips to room temperature before use.
2. Prepare 1X Wash Buffer by diluting 20X wash buffer (included in each PathScan^® Sandwich ELISA Kit) in dH₂O.
3. 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
   • 1µg/ml leupeptin

B. 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.

C. Test Procedure

1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed and stored at 4°C immediately.
2. Add 100 µl of Sample Diluent (supplied in each PathScan^® Sandwich ELISA Kit, blue color) to a microcentrifuge tube. Transfer 100 µl of cell lysate into the tube and vortex for a few seconds. (Sample applied to the well can be diluted 1:1 when the suggested cell lysis buffer is used for cell extraction.) Individual datasheets for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
3. Add 100 µl of each diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hours at 37°C. Alternatively, the plate can be incubated overnight at 4°C, which yields the best detection of target protein.
4. Gently remove the tape and wash wells:
   1. Discard plate contents into a receptacle.
   2. Wash 4 times with 1X Wash Buffer, 200 µl each time for each well.
   3. 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.
   4. Clean the underside of all wells with a lint-free tissue.
5. Add 100 µl of Detection Antibody (green color) to each well. Seal with tape and incubate the plate for 1 hour at 37°C.
6. Repeat wash procedure as in Step C4.
7. Add 100 µl of HRP-Linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 minutes at 37°C.
8. Repeat wash procedure as in Step C4.
9. Add 100 µl of TMB Substrate to each well. Seal with tape and incubate the plate for 10 minutes at 37°C or 30 minutes at 25°C.
10. Add 100 µl of STOP Solution to each well. Shake gently for a few seconds.

NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

1. Read results.
   1. Visual Determination — Read within 30 minutes after adding STOP Solution.
   2. Spectrophotometric Determination — Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 minutes after adding STOP Solution.