Anisha Patel COSMOS UC Davis Cluster 1: Biotechnology

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6. Grind approximately five grams of frozen cell tissue in a mortar.i. Add liquid nitrogen, grind slowly as it boils off. Repeat grinding step three times.7. Add a small amount of liquid N2, and swirl to break loose the ground tissue.8. Transfer ground tissue to a 4 degrees C mortar and add 8 mL of Tris extraction bufferi. Grind until tissue and buffer are thoroughly mixed.ii. Prepare Tris Extraction Buffer: 40 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 2%PVPP, 125 U endonuclease/mL, 1 mM PMSF9. Pour mixture into 13 mL centrifuge tube, vortex, and sonicate.i. Vortex for 1 minute, set on ice for 1 minute.ii. Sonicate for 10 pulses (Each Pulse = 2 sec). Set on ice for 3 minutes.iii. Repeat (a) and (b) for a total of two times.10. Centrifuge at 5,000 g for five minutes (6,500 rpm) to pellet debris and recover the supernatant.11. Precipitate protein by adding 4.33 mL of 37.5% TCA plus 1.0% β-mercaptoethanol,bring to a final volume of 13 mL using 37.5% TCA, incubate on ice for 45 minutes,and centrifuge at 14,000 g for 15 min.12. Wash protein pellet 3 times in 80% acetone containing0.05% β-mercaptoethanol.i. Break up protein pellet using small metal spatula.ii. Sonicating in a water bath for five minutes will help disrupt the pellet*.13. Air dry protein pellet in laminar flow hood until all the liquid is gone.
PART 114. Add 10-15 µl of Laemmli buffer to a microtube.15. Add beta mercaptoethanol. (5% of total volume).16. Add a protein sample volume that will contain 100 – 150 µg of protein to the tube withthe other reagents.17. Heat the microtubes at 70 degrees Celsius for 2 minutes, then place on ice18. Pour SDS Buffer into the Western Blot tank.19. Position the acrylamide gels in the gel holder assembly and immerse into the tank.20. Fill the inner compartment (between the two gels) with SDS Buffer.21. Carefully load the samples in the wells.22. Place the lid on the tank and plug it into the power source (100V).23. Run the apparatus at 100V until the samples have passed the stacking gel.24. Turn the voltage up to 160V and allow the samples time to separate; use a pre-stainedmolecular weight marker to determine the end-point of the electrophoresis.25. Cut filter paper in approximately7 x 20 cm pieces.26. Cut PVDF membrane to 7 x 20 cm.27. Pre wet the PVDF membrane using 100% methanol for 10 seconds and immerse indH2O.
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Anisha Patel COSMOS UC Davis Cluster 1: Biotechnology

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