1X Cell Extraction Buffer: Prepare by diluting FastScan™ ELISA Cell Extraction Buffer (5X) #69905 and FastScan™ ELISA Cell Extraction Enhancer Solution (50X) #25243* to 1X with deionized water. This buffer can be stored at 4°C for short-term use (1-2 weeks). To make 10 mL 1X Cell Extraction Buffer, combine 7.8 mL deionized water, 2 mL FastScan™ ELISA Cell Extraction Buffer (5X), and 200 μL FastScan™ ELISA Cell Extraction Enhancer Solution (50X). Alternatively, Enhancer Solution may be added to the Cell Extraction Buffer after extraction of cells or tissue. When using the 1X Cell Extraction Buffer as a sample diluent for the assay, it is recommended to equilibrate it to room temperature prior to use.
*IMPORTANT: The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.
NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).
Preparing Cell Lysates
For adherent cells
1. Aspirate media when the culture reaches 80–90% confluence.
2. Remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 mL ice-cold 1X Cell Extraction Buffer (recommended to supplement with protease inhibitors and additional phosphatase inhibitors as needed) to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
5. Sonicate lysates on ice.
6. Microcentrifuge for 5 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
1. Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml.
2. Wash once with ice-cold 1X PBS.
3. Cells harvested from 50 mL of growth media can be lysed in 2.0 mL of 1X Cell Extraction Buffer (recommended to supplement with protease inhibitors and additional phosphatase inhibitors as needed).
4. Sonicate lysates on ice.
5. Microcentrifuge for 5 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.
FastScan™ ELISA Cell Extraction Buffer (5X) should be stored at 4ºC.
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