Non-Radioactive In vitro Kinase Assay - MPF and MAPK specific (Mar/04/2009 )
Been trying to track down if anyone has come up with an alternative to radioactivity for the assay conditions. I have a basic protocol that want to use (see below) but was wondering if maybe a biotin or dig labeled ATP could be substituted for 32P labeled. Justed wanted to see what others thought.
In Vitro Kinase Activities Assay: Assays will be performed as described in Tian et al. (2002). Briefly, Groups of 10 oocytes or presumed zygotes will be lysed in cold 4 μL PBS containing 6.4 mM EDTA, 10 mM sodium floride (NaF), 100 mM sodium orthovanadate (Na3VO4) then stored at -80°C until assay. Each sample will mixed with 6 μL of H1 kinase buffer (45 mM β-glycerophosphate, 12 mM EGTA, 12 mM MgCl2, 0.8 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 20 μg/mL leupetin, 40 μg/mL aprotinin, 10 µg/ml pepstatin, and 500 nmol/l cAMP dependent protein kinase inhibitor). The samples will be incubated at 37°C for 15 min. Then to 10μL of each sample (performed in duplicate) 8 μL of H1 kinase buffer with 2 mg/mL histone H1 (Type III-S, Sigma, St. Louis, MO), 1 mg/mL myelin basic protein (Sigma), 250 μCi/mL <γ-32P>ATP (GE Healthcare, Piscataway, NJ). The reaction will be incubated for 30 min at 37°C and then terminated by the addition of 5 µl of double-strength electrophoresis sample buffer. Included in assay will be controls consisting of all components except cellular lysate. Samples will be denatured (95°C for 5 min) prior to loading onto 8-15% linear gradient SDS polyacrylamide gel. Gels will be dried then exposed to Kodak X-ray films to be scanned to quantify kinase activity. Multiple exposures will be performed to avoid saturation of exposed film.
if you have a specific antibody for phosphorylated substrate vs unphosphorylated,, you can use that in a western blot of your reac tion products.
If your phosphorylated substrate runs higher than your unphosphorylated one, you can detect that by just a mol. wt difference 9assuming you are phosphorylating a protein).