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        CAT ASSAY (liquid phase)

        互联网

        1210

        1. Transfer cells to a 15 ml tube.

        2. Add 5 ml TBS- to flasks, shake & pour into tubes.

        3. Spin down the cells @ 1K RPM for 5'.

        4. Resuspend in 1 ml TBS-.

        5. Transfer to 1.5 ml tubes.

        6. Spin down @ 14K RPM for 2-3', remove supernatant.

        7. Resuspend in 100 µl buffer I (250 mM Tris pH 7.8/5 mM EDTA).

        8. Freeze-thaw 3x (10'freeze-10'thaw).

        9. Spin down debris @ 14K RPM for 5'.

        10. Transfer 100 µl supernatant to a new set of tubes.

        11. Set new set of tubes and add 10 µl of supernatant and 50 µl buffer I in each.

        12. Add 20 µl of 5mM chloramphenicol in buffer I(or 20 µl of buffer I for a blank).

        13. Incubate 5 min @ 37℃

        14. Make the following mix:

        0.75 µl of 4mM acetyl CoA

        2.0 µl 750 µM HCl (10X)

        16.25 µl H2 O

        1.0 µl 3H acetyl-CoA

        (Times number of samples)

        15. Add 20 µl of mix into each tube (reaction & blank).

        16. Incubate for 2 hrs @ 37℃.

        17. Add 500 µl of toluene to each tube.

        18. Vortex for 10 sec.

        19. Spin down for 1 min.

        20. Remove 400 µl into scintillation tubes.

        21. Add 10 ml of scintillation liquid.

        22. Count it for 2 min.

        (total cpm's in reaction is 210000-230000)

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