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
Dilute the extract at least five-fold in H1 Kinase Buffer.
Add a sufficient amount of antibody that recognizes the tag used in constructing the tagged Mos protein (see Tip#2).
Incubate on ice for 1 hr (see Tip#3).
Protein A Sepharose Antibody Binding Protocol
Equilibrate Protein A Sepharose 6MB beads (Pharmacia) with H1 Kinase Buffer by suspending the beads in the buffer (see Tip #4).
Centrifuge for 30 sec or less in a microcentrifuge to pellet the beads.
Remove the supernatant and repeat Steps #C1 and #C2 two more times.
Resuspend the beads in H1 Kinase Buffer to give a 50% slurry of beads.
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).
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.
Centrifuge the antibody/extract solution at maximum speed for 2 min in a microcentrifuge to pellet any insoluble material (see Tip#6).
Add the supernatant to the Protein A Sepharose slurry.
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.
Centrifuge the samples for 30 sec or less in a microcentrifuge to pellet the beads.
Wash the beads by resuspending in Wash Buffer. Fill the tube three-quarters full.
Centrifuge the samples for a 30 or less in a microcentrifuge to pellet the beads and remove the supernatant.
Resuspend the beads in Wash Buffer again and transfer the beads to a new tube.
Rinse the old tubes with more Wash Buffer and add that wash to the new tubes.
Centrifuge the beads for 30 sec or less in a microcentrifuge to pellet the beads and remove the supernatant.
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
D) (or go to Section E)
Wash the beads one or two times in H1 Kinase Reaction Buffer.
Aspirate off all of the supernatant.
Add 10 μl of H1 Kinase Reaction Buffer containing 20 μM ATP and 5 μCi γ-[32P]-ATP (CAUTION! see Tip#7).
Incubate the reaction for 10 min at 30°C.
Stop the reaction by adding 10 μl of 2X Sample Buffer.
Load 8 μl of the sample on a 15% Polyacrylamide gel (see Protocol on SDS-PAGE).
Run the gel and process it for autoradiography (see Protocol on Autoradiography).
Polyacrylamide Gel Analysis of Immunoprecipitation Protocol
E) (see Tip #8)
Resuspend the beads in 1X Sample Buffer.
Boil the samples for 90 sec.
Centrifuge the samples briefly in a microcentrifuge.
Load the samples onto an SDS-Polyacrylamide gel (see Protocol on SDS-PAGE).
Electrophorese the gel.
Dry the gel on a gel dryer.
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
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
Glycerol 2-Phosphate, Sodium Salt
Protein A Sepharose
1. Treat the extract with nuclease to increase translational efficiency.
2. To keep the buffer more or less equivalent to H1 Kinase Buffer, it is best not to use a volume of antibody that exceeds one-fifth the total incubation volume.
3. This length of time probably greatly exceeds the time needed for antibody/antigen binding.
4. Pre-equilibrate the Protein A Sepharose beads in the last 5 to 10 min of the antibody/antigen incubation.
5. The volume of beads needed to bind all the available antibody is much lower than that needed for the kinase assay. The beads have a very high capacity for IgG and affinity-purified antibodies have relatively low concentrations of antibody. Therefore, 10 μl of packed beads is usually enough to bind 200 μl of affinity-purified antibody.
6. This is the last chance to remove contaminating insoluble material.
7. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
8. If the Protein A Sepharose Beads 6MB Beads were mixed with a 35S-labeled extract and are to be analyzed for immunoprecipitation of 35S-labeled proteins, the beads can be processed for SDS-PAGE and autoradiography instead of a kinase assay.
References for Kinase Assay Protocol
This is based on the protocol by Andrew Murray, Harvard University.