Trident Membrane Protein Extraction Kit

Transcription

1 Cat. No. Size Shelf life GTX / 20 tests 12 months at the appropriate storage temperatures (see below) Contents Component Storage Amount for 5 tests Amount for 20 tests Buffer A -20 o C 2.5 ml 10 ml Buffer B -20 o C 1 ml 4 ml Protein extraction filter cartridges Room temperature 5 20 Collection tubes with caps Room temperature 5 20 Tissue dissociation beads Room temperature 0.5 g 2 g Plastic rod Room temperature 1 1 Description This kit is for rapid extraction of native total membrane proteins from cultured mammalian cells or tissues. Simple and user-friendly filter cartridge-based technology Broad range of starting cells (1-50 million cells / sample) or fresh/frozen tissue (10-30 mg) No detergent or EDTA No requirement for Dounce homogenizer or tissue blender Kit protocol can be completed in less than 45 minutes (min) High yield Note 1. Read the entire protocol carefully. 2. Before starting, thaw Buffer A and Buffer B (if needed) completely, invert the bottles several times and place on ice. Insert the filter cartridges in collection tubs and pre-chill on ice. Pre-chill 1X PBS on ice. 3. All centrifugation steps should be performed at 4 (either in a cold room or in a refrigerated microcentrifuge). 4. To study protein phosphorylation, phosphatase inhibitors should be added to Buffer A prior to use. The use of protease inhibitor cocktails is optional. 5. This product is for research use only. Materials required but not supplied: 1X PBS Vortexer Table-top refrigerated microcentrifuge High-salt Extraction Buffer (20 mm HEPES, ph 7.9, 1.5 mm MgCl 2, 0.42 M NaCl, 0.2 mm EDTA 25% (v/v) Glycerol) 1

2 Protocol A. Extraction of Total Membrane Proteins 1. For cultured cells, collect 1 x x 10 6 cells by low-speed centrifugation ( x g, 5 min). For tissue samples, start from step 2. Note: If plasma membrane proteins are to be extracted from cultured cells, we recommend starting with X 10 6 cells. Please see below Protocol D for details. Wash cells once with cold 1X PBS. Aspirate supernatant completely and resuspend the pellet in cold Buffer A (200 µl for a starting cell number less than 5 X 10 6 cells and 500 µl for a starting cell number greater than 5 X 10 6 cells). Incubate the cell suspension on ice for 5-10 min. Vortex the tube vigorously for seconds. Immediately transfer the cell suspension to a pre-chilled filter cartridge, and proceed to step For tissue samples, place a piece of fresh tissue (10-30 mg) or frozen tissue (20-30 mg) in a pre-chilled filter cartridge. Add 200 µl Buffer A to the filter and grind the tissue with the plastic rod for at least 1 min by pushing the tissue against the surface of the filter repeatedly with a twisting motion. Note: If you are working with skeletal or cardiac muscle tissue, it is recommended to add mg tissue dissociation beads to the filter prior to grinding. Add an additional 300 µl of Buffer A to the same filter cartridge, mix by pipetting up and down several times and incubate the tube on ice with cap open for 5 min. Go to step 3. Note: The presence of a small amount of non-homogenized tissue will not affect the quality of the sample. The plastic rod is reusable: after use, wipe it with 75% alcohol or rinse it with distilled water. 3. Cap the filter cartridge and centrifuge at 14,000 rpm (16,000 xg) for 30 seconds. Optional: For cultured cells we recommend to resuspend the pellet in the collection tube from step 3, transfer the cell suspension back to the same filter and spin at 14,000 rpm (16,000 xg) for 30 seconds. Re-passing the cells through the filter can increase the yield by 20% - 30%. 4. Discard the filter and resuspend the pellet by vigorously vortexing for 10 seconds. The following procedure separates total cellular components into four fractions: nuclei, cytosol, organelles and plasma membrane. 2

3 5. Centrifuge at 3,000 rpm (700 xg) for 1 min. The pellet contains intact nuclei. If nuclear protein is to be extracted, proceed to step 6. If cytosolic protein is to be extracted, please keep supernatant and proceed to step 9. B. Extraction of Nuclear Proteins 6. Resuspend the crude nuclei pellet (5X nuclei pellet) on High-salt Extraction Buffer containing protease inhibitor cocktail. Note: if you want to verify for phosphorylation protein, we would suggest the phosphatase Inhibitors with extraction buffer. 7. Mix well by vortex mixer and then spin down the buffer. After incubation on ice for 10 mins, the sonicator had speed of 70ampl and turned on for 3 seconds and off for 3 seconds maintaining the same duty cycle with 3 times. Finally, centrifuge for 5 minutes at 14,000 rpm (16,000 xg). 8. Transfer the supernatant to fresh tube. Aliquots and store at -80 C. C. Collection of cytosolic Proteins 9. Transfer the supernatant of Step 5 to a fresh 1.5 ml microcentrifuge tube and centrifuge at 4 C for min at 14,000 rpm (16,000 xg) (a longer centrifugation time will increase yield). The supernatant is the cytosolic fraction. Remove the supernatant and save the pellet. The pellet is the total membrane protein fraction including organelle and plasma membranes. 10. If plasma membrane protein is to be extracted, proceed to step 11. If not, store the pellet at -70 o C or dissolve it in the detergent-containing buffer of your choice. Don t freeze total membrane protein fraction if further isolation of plasma membrane proteins is desired. The typical yield of the total membrane protein fraction is µg/sample. D. Extraction of Plasma Membrane Proteins 11. Resuspend the total membrane protein fraction from step 9 in 200 µl Buffer B by repeatedly pipetting up and down or vortexing. Centrifuge at 10,000 rpm (7,800 xg) for 5 min at 4 o C. The pellet contains organelle membrane proteins. 3

4 12. Carefully transfer the supernatant to a fresh 2.0 ml microcentrifuge tube and add 1.6 ml cold 1X PBS. Mix by inverting the tube several times. Centrifuge at 14,000 rpm (16,000 xg) for min. (a longer centrifugation time will improve yield). Discard the supernatant and save the pellet. This is the extracted plasma membrane protein fraction. Typically µg of plasma membrane protein is obtained. The pellet of plasma membrane proteins can be dissolved in µl of the detergent-containing buffer of your choice (such as 0.5% Triton X-100 in PBS). Troubleshooting Problem Low protein yield Low protein activity Retention of cell lysate in protein filter cartridge after 30 seconds of centrifugation Solution Increase starting cell numbers. Increase incubation time to 10 min (steps 1 or 2). Keep lysate cold/add protease inhibitors. Reduce amount of starting material or increase centrifugation time to 2 min. 4

5 5