Found 1 discussion for this experiment
2 years ago
2 years ago by Hollie Fowler
I am using 8M Urea to lyse my cells but after centrifugation my pellet is very viscous. This causes me trouble when I try to collect the supernatant. Is there any way to avoid it and do you think it will have an effect on the concentration of my protein?
Found 2 matching solutions for this experiment
|- There is a previous cell lysate step.|
|- Total cell lysate is subjected to ultracentrifugation (435,700 × g, 1 h, 4°C) to pellet membrane vesicles.
- Membrane pellets are rinsed in 1.0 ml of HN buffer with protease inhibitors, reisolated by ultracentrifugation, and suspended in 200 to 400 μl of HN buffer plus complete protease inhibitor cocktail with the aid of a coated homogenizer.
|- Protein precipitation is performed following Trichloroacetic Acid Solution, MP Biomedicals™ protocol (Fisher Scientific).|
|- Previous isolation of membrane proteins with Gyros IncSupplier Diversity Partner REXXIP HN BUFFER 25 ML PER VI DFS Item (Fisher Scientific).|
|- Membrane proteins are precipitated by the addition of 8 volumes acetone (Fisher), 1 volume of a saturated trichloroacetic acid solution (Fisher). Incubation: −20°C for 45 min. After, are subjected to centrifugation (16,000 × g, 20 min, 4°C), rinsed in 80% cold acetone in distilled water, air dried, and solubilized.|