In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translation Protocol of Xenopus Mos Kinase

•  Translate tagged Mos in a CSF extract (see Protocol on CSF Extract Preparation and see Tip #1).

Antibody to Antigen Binding

1. Dilute the extract at least five-fold in H1 Kinase Buffer.
2. Add a sufficient amount of antibody that recognizes the tag used in constructing the tagged Mos protein (see Tip#2).
3. Incubate on ice for 1 hr (see Tip#3).

Protein A Sepharose  Antibody Binding Protocol

1. Equilibrate Protein A Sepharose 6MB beads (Pharmacia) with H1 Kinase Buffer by suspending the beads in the buffer (see Tip #4).
2.  Centrifuge for 30 sec or less in a microcentrifuge to pellet the beads.
3. Remove the supernatant and repeat Steps #C1 and #C2 two more times.
4.  Resuspend the beads in H1 Kinase Buffer to give a 50% slurry of beads.
5. To determine how much Protein A Sepharose to use, consider how many kinase reactions will be carried out with each immunoprecipitate. It is important that each of the kinase reaction tubes receive about 10 μl of packed beads. Thus, if an immunoprecipitation is to be split 4 ways, use 80 μl of a 50% suspension of Protein A Sepharose for the Protein A Sepharose/antibody binding step. For these calculations, it is not important to consider the binding affinity of the Protein A Sepharose (see Tip#5).
6. Aliquot the buffer-equilibrated Protein A Sepharose beads into microcentrifuge tubes. Use 0.6 ml tubes if the volume will be less than 0.5 ml; otherwise, use 1.5 ml tubes.
7.  Centrifuge the antibody/extract solution at maximum speed for 2 min in a microcentrifuge to pellet any insoluble material (see Tip#6).
8. Add the supernatant to the Protein A Sepharose slurry.
9.   Incubate for 30 min at room temperature with end-over-end mixing. To ensure good mixing during the incubation, fill the tubes three-quarters full so that an air bubble moves up and down the tube, continuously mixing the contents.
10.  Centrifuge the samples for 30 sec or less in a microcentrifuge to pellet the beads.
11.   Wash the beads by resuspending in Wash Buffer. Fill the tube three-quarters full.
12.   Centrifuge the samples for a 30 or less in a microcentrifuge to pellet the beads and remove the supernatant.
13.   Resuspend the beads in Wash Buffer again and transfer the beads to a new tube.
14.   Rinse the old tubes with more Wash Buffer and add that wash to the new tubes.
15.   Centrifuge the beads for 30 sec or less in a microcentrifuge to pellet the beads and remove the supernatant.
16.   Wash again with Wash Buffer and centrifuge the beads for 30 sec or less in a microcentrifuge and remove the supernatant.

Kinase Reactions on Immunoprecipitated Material

1. Wash the beads one or two times in H1 Kinase Reaction Buffer.
2. Aspirate off all of the supernatant.
3. Add 10 μl of H1 Kinase Reaction Buffer containing 20 μM ATP and 5 μCi γ-[32P]-ATP (CAUTION! see Tip#7).
4. Incubate the reaction for 10 min at 30°C.
5. Stop the reaction by adding 10 μl of 2X Sample Buffer.
6. Load 8 μl of the sample on a 15% Polyacrylamide gel (see Protocol on SDS-PAGE).
7. Run the gel and process it for autoradiography (see Protocol on Autoradiography).

Polyacrylamide Gel Analysis of Immunoprecipitation Protocol

1. Resuspend the beads in 1X Sample Buffer.
2. Boil the samples for 90 sec.
3. Centrifuge the samples briefly in a microcentrifuge.
4. Load the samples onto an SDS-Polyacrylamide gel (see Protocol on SDS-PAGE).
5. Electrophorese the gel.
6. Dry the gel on a gel dryer.
7. Detect kinase activity by autoradiography (see Protocol on Autoradiography).

Buffers for Kinase Assay Protocol

H1 Kinase Buffer		1 mM DTT (to retain activity of the protein) 20 mM EGTA 15 mM MgCl2 80 mM Glycerol 2-Phosphate, Sodium Salt, pH 7.4
Sample Buffer (2X)		2 mM DTT Add DTT just before use 20% (v/v) Glycerol 100 mM Tris-Cl, pH 6.8 0.2% (w/v) Bromophenol Blue 4% (w/v) SDS
H1 Kinase Reaction Buffer		15 mM MgCl2 1 mM DTT 20 mM HEPES, pH 7.4 150 mM NaCl
Wash Buffer		1 mM DTT 0.1% (v/v) Triton-X 100 5 mM EGTA 50 mM Tris-Cl, pH 7.4 5 mM NaF 250 mM NaCl

  Materials Required for the Method

• Triton-X 100
• Bromophenol Blue
• Glycerol
• Tris
• Magnesium Chloride
• I3-[³²P]-ATP
• DTT
• Antibody
• EGTA
• ATP
• Sodium Fluoride
• Glycerol 2-Phosphate, Sodium Salt
• Sodium Chloride
• Protein A Sepharose
• SDS
• HEPES

Protocol Tips

References for Kinase Assay Protocol