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Red Loading Buffer Pack #7723
Description
The Red Loading Buffer Pack is used to lyse cells for Western blotting analysis
Directions for Use
(a) Prepare fresh 3X Reducing SDS Loading Buffer by adding 1/10 volume 30X Reducing Agent to 1 volume of 3X SDS Loading Buffer.
(b) Aspirate media from cultures; wash cells with 1X PBS; aspirate.
(c) Lyse cells by adding 1X SDS Sample 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.
(d) Sonicate for 10-15 seconds to shear DNA and reduce sample viscosity.
(e) Heat a 20 µl sample to 95-100°C for 5 minutes; cool on ice.
(f) Microcentrifuge for 2-5 minutes.
(g) Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
Solutions and Reagents
Quantity 8 ml 3X Blue Loading Buffer and 1 ml 30X Reducing Agent
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.)
Application References
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