Product Pathways - Apoptosis / Autophagy
Chaps Cell Extract Buffer (10X) #9852
Description
Chaps Cell Extract Buffer can be used to lyse cells under nondenaturing conditions and is recommended for the preparation of cytoplasmic cell lysates to be used with our caspase signaling pathway antibodies (see Companion Products).
Directions for Use
- Buffer should be prepared immediately before use.
- Immediately before dilution, invert the Chaps Cell Extract Buffer several times to suspend all buffer components.
- Add 1/10 volume of Chaps Cell Extract Buffer to 9/10 volume of Milli-Q water or equivalently purified water.
- Add DTT to final concentration of 5 mM (1:200 dilution) and PMSF* to final concentration of 1 mM.
*reagent not included
Sample Preparation Using Chaps Cell Extract Buffer
- Treat cells by adding fresh media containing regulator for desired time.
- Aspirate media from cultures; wash cells three times with PBS; aspirate. Scrape cells into PBS and spin down to pellet.
- Lyse cells by adding Chaps Cell Extract Buffer (1 volume of cell pellet). Resuspend cells in the buffer, freeze and thaw three times, centrifuge lysate at 14,000 rpm. Keep the supernatant and discard the pelleted cell debris.
- Add SDS Sample Buffer and heat sample to 95–100°C for 5 minutes; cool on ice.
- Microcentrifuge for 5 minutes.
- Load 5 µ| onto SDS-PAGE gel (10 cm x 10 cm).
Store Chaps Cell Extract Buffer and 200X dithiothreitol (DTT) at -20°C.
Solutions and Reagents
- 10X Chaps Cell Extract Buffer (5 ml)
1X concentration: - 50 mM Pipes/HCI (pH 6.5)
- 2 mM EDTA
- 0.1% Chaps
- 20 µg/ml Leupeptin
- 10 µg/ml Pepstatin A
- 10 µg/ml Aprotinin
- 200X DTT (0.25 ml)
1X concentration: - 5 mM DTT
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
