News from the Bench
Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.
Research More. Spend Less.
Spend $650 or more and get 20% off*
(*Offer valid in the US only. Expires June 30, 2017)
To Purchase # 7723S
Find answers on our FAQs page.
- Additional protein information
- Analytical tools
Red Loading Buffer Pack #7723
Gallery: Red Loading Buffer Pack #7723
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).Solutions and Reagents:
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.)Storage: Store at Room Temperature and -20°C.3X Red Loading Buffer Pack: 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.)
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.