|3X Red Loading Buffer||8 ml|
|30X Reducing Agent (1.25 M DTT)||1 ml|
The Red Loading Buffer Pack is used to lyse cells for western blotting analysis.
1. Prepare fresh 3X Reducing SDS Loading Buffer by adding 1/10 volume 30X DTT Reducing Agent to 1 volume of 3X SDS Loading Buffer.
2. Dilute 3X SDS Loading Buffer to a 1X solution using ddH2O. This product supplies enough 3X material to make 24ml of 1X solution.
3. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
4. Lyse cells by adding 1X SDS Loading Buffer (100 µl per well of 6-well plate or 500 µl per plate of 10 cm2 plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
5. Sonicate for 10-15 seconds to shear DNA and reduce sample viscosity.
6. Heat a 20 µl sample to 95-100ºC for 5 minutes; cool on ice.
7. Microcentrifuge for 2-5 minutes.
8. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
Quantity 8 ml 3X Red Loading Buffer and 1 ml 30X Reducing Agent
3X Red Loading Buffer Pack: 187.5 mM Tris-HCl (pH 6.8 at 25ºC), 6% (w/v) SDS, 30% glycerol and 0.03% (w/v) phenol red. (Store at room temperature.)
30X Reducing Agent: 1.25 M dithiothreitol (DTT) (Store at -20ºC.)
Store at Room Temperature and -20°C.3X Red Loading Buffer: 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% (w/v) SDS, 30% glycerol and 0.03% (w/v) phenol red. (Store at room temperature.)30X Reducing Agent: 1.25 M dithiothreitol (DTT) (Store at -20°C.)
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.
To get local purchase information on this product, click here.