- 96-Well aluminum block for the PCR-plate to keep it cold on
ice during the IP procedure and warm on a heating block
during the kinase reaction (Fig.1A). - Rotation apparatus able to hold the “sandwich device” with the
PCR-plate(s). Should be placed at 4C. - 96-Well plate swing-out rotor insert for centrifuge.
- 6Assay buffer: 240 mM HEPES, pH 7.0, 480 mM NaCl.
This buffer is stored at 4C. - 3Assay buffer: 120 mM HEPES, pH 7.0, 240 mM NaCl.
This buffer is stored at 4C. - Adjustable eight-channel pipette going up to 200μl.
- Homemade eight-channel suction device (Fig.1C). Used for
aspiration of wash buffer. An eight-channel pipette could be
used instead.
2.2 Kinase Reaction 1. Kinase reaction buffer (30μl per sample): 40 mM HEPES,
pH 7.0, 80 mM NaCl, 833μM DTT, 200μM AMP, 100μM
AMARA peptide (AMARAASAAALARRR), 5 mM MgCl 2 ,
200 μM ATP, 33μCi/ml^33 Pγ-labeled ATP (seeNote 4).
- Heating block for the PCR-plate (30C) (seeNote 5).
- Repeater pipette with options to dispense 10 and 30μl.
- Phosphoric acid (1% (v/v) solution).
- Eight-channel pipette in the range of 20μl.
- Whatman™Grade P81 Ion Exchange Cellulose Chromatog-
raphy Paper. - Acetone.
- Ultima Gold™scintillation cocktail.
- Pico Prias Vials (6 ml).
- Phosphor imager with storage phosphor screens.
- Liquid Scintillation Analyzer.
3 Methods
We strongly recommend to homogenize the muscle tissue as
described in Chapter 13, as we have seen that muscles homoge-
nized in a standard RIPA buffer (Sigma R0278) or Rac1 lysis buffer
(Cytoskeleton Inc. BK126) have no AMPK activity at all (unpub-
lished data).3.1 Immuno-
precipitation
(IP) Procedure
- Prepare a list of your samples, so you will know how much of
each buffer and reagent is necessary for the entire assay. The
following will be an example of an assay with 100 samples
Kinase Activity of Specific Heterotrimers 219