PathScan® Sandwich ELISA Protocol (Chemiluminescent)
NOTE: Refer to product-specific datasheets for assay incubation temperature. This chemiluminescent ELISA is offered in low volume microplate. Samples and reagents only require 50 µl per microwell.
A. Solutions and Reagents
NOTE: Prepare solutions with purified water.
- Microwell strips: Bring all to room temperature before use.
- 1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in purified water.
- 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 1 mM phenylmethylsulfonyl fluoride (PMSF) immediately before use.
- 1X PBS: (20X Phosphate Buffered Saline #9808).
- 20X LumiGLO® Reagent and 20X Peroxide (#7003).
B. Preparing Cell Lysates
For adherent cells
- Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
- Remove media and rinse cells once with ice-cold 1X PBS.
- Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
- Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
- Sonicate lysates on ice.
- Microcentrifuge for 10 min (x14,000 rpm) 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.
For suspension cells
- Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
- Collect cells by low speed centrifugation (~1200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
- Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X Cell Lysis Buffer plus 1 mM PMSF.
- Sonicate lysates on ice.
- Microcentrifuge for 10 min (x14,000 rpm) 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
- 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.
- Cell lysates can be undiluted or diluted with Sample Diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
- Add 50 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at room temperature. Alternatively, the plate can be incubated overnight at 4°C.
- Gently remove the tape and wash wells:
- Discard plate contents into a receptacle.
- Wash 4 times with 1X Wash Buffer, 150 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 dry completely at any time.
- Clean the underside of all wells with a lint-free tissue.
- Add 50 µl of Detection Antibody (green color) to each well. Seal with tape and incubate the plate at room temperature for 1 hr.
- Repeat wash procedure (Section C, Step 4).
- Add 50 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate at room temperature for 30 min.
- Repeat wash procedure (Section C, Step 4).
- Prepare Detection Reagent Working Solution by mixing equal parts 2X LumiGLO® Reagent and 2X Peroxide.
- Add 50 µl of the Detection Reagent Working Solution to each well.
- Use a plate-based luminometer to measure Relative Light Units (RLU) within 1–10 min following addition of the substrate.
- Optimal signal intensity is achieved when read within 10 min.
posted November 2009
posted September 2012
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