|3X Red Loading Buffer||8 ml|
|30X Reducing Agent (1.25 M DTT)||1 ml|
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 Agent3X 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.)
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