Kinase assay with Cdk immunoprecipitates from embryos

1. immunocomplexes of Cdk

• collect embryos, dechorionate, determine the weight and freeze in liquid nitrogen. Store at -80¡C
• homogenise the embryos in IP buffer (ca 1 ml per 500 embryos-5mg)
• spin 10 min at 13k
• extract the supernatant with 1,1,2 Trichlorotrifluoroethan
• spin with UZ for 15 min
• add a-Cdk1 antibody (ca 1 ul serum #148 or 1 ug IgG per 1000 embryos) to the supernatant
• mix, incubate for 1h at 4¡C
• add proteinA-sepharose beads (50 ul per 1000 embryos, washed in IP buffer)
• incubate on wheel for 1h at 4¡C
• wash the beads 5 x with IP buffer
• wash twice with 1x kinase buffer
• distribute corresponding amounts of the beads to reaction tubes

2. kinase reaction

• prepare 1x kinase buffer (for washing of the beads, see above)
• prepare 2x kinase buffer
• dilute g-32P-ATP to 2 uCi/ul
• set up the reaction mix on ice
• start the reaction by addition of the mix to the beads
• incubate for 20 min at 25¡C
• stop the reaction by adding LŠmmli buffer
• boil for 3 min

2 ul 5x buffer with DTT and ATP, ATP is 10x concentrated
1 ul g-ATP (2 uCi/ul, final 2 uCi per reaction)
2 ul Histon H1 (30 uM, final 3 uM)
X ul water
10 ul suspension of beads (in 1x kinase buffer without ATP)
----
20 ul

3. analysis by SDS-PAGE

• analyse the protein mix by a 12% gel with SDS-PAGE
• dissemble the apparatus, place the gel on a whatman filter
• stain and destain the gel or only incubate with destaining solution for 30 min
• cover the gel with a foil
• dry the gel onto the whatman filter in a gel dryer (1h, 80¡C)
• expose a phospho imager screen overnight

4. Buffer, solutions

1x Kinase buffer

25 mM Hepes/NaOH, pH 7,4
10 mM MgCl2
1 mM DTT
10 uM ATP

IP buffer

50 mM Hepes/NaOH pH7,5
150 mM NaCl
0,5% TritonX-100
10% glycerol

protein A sepharose

radioactive ATP