Buffers
Blue Loading Buffer Pack #7722
Description
The Blue Loading Buffer Pack is used to lyse cells for Western blotting analysis
Directions for Use
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 Blue Loading Buffer and 1 ml 30X Reducing Agent
3X Blue Loading Buffer Pack: 187.5 mM Tris-HCl (pH 6.8 at 25ºC), 6% (w/v) SDS, 30% glycerol and 0.03% (w/v) bromophenol blue. (Store at room temperature.)
30X Reducing Agent: 1.25 M dithiothreitol (DTT) (Store at -20ºC.)
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!
For Research Use Only. Not For Use In Diagnostic Procedures.
