Kinase reactions were carried out in two steps. First, Sf9-purified 0.2 µg of human His-Plk1 (Sino Biological) was preincubated in a kinase cocktail [50 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 2 mM dithiothreitol, 2 mM EGTA, and 27 mM p-nitrophenylphosphate] in the presence of either Allopole-A or p-13mer PBIP peptide for 20 min at 25 °C. The samples were then added with 1 µg of dephosphorylated casein (Sigma) and 1.25 µM of ATP (1.25 µCi of [γ32P] ATP, 1Ci = 37 GBq) and reacted for additional 30 min at 30 °C. The reactions were terminated by the addition of 5× Laemmli SDS sample buffer, separated by 10% SDS-PAGE, and analyzed by autoradiography. To quantify the γ32P signals, appropriate bands were excised from dried SDS-PAGE gels and analyzed by a liquid scintillation counter (Beckman model LS 6500).
Dephosphorylated casein
Manufactured by Merck Group
Dephosphorylated casein is a laboratory reagent used as a blocking agent in various biochemical and immunological assays. It is derived from casein, a protein found in milk, and has had its phosphate groups removed through a dephosphorylation process. Dephosphorylated casein is commonly used to reduce non-specific binding in techniques such as Western blotting, ELISA, and immunohistochemistry, thereby improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using dephosphorylated casein
1
Plk1 Kinase Assay Protocol
2
CK1δ Kinase Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pCK1δ plasmids were pCS2-6Myc-CK1δ (V2418). PF670462, PF4800567 and staurosporine were purchased from Tocris Bioscience. Dephosphorylated casein was purchased from Sigma Aldrich. Cell lines were from American Type Culture Collection (ATCC), USA.
3
Kinase Assays for SRPK1 and CK2 Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human SRPK1
and a fragment of the N-terminal domain of turkey Lamin B Receptor
(LBR) comprising amino acids 62–92 (LBRNt(62-92)) were subcloned
into pGEX-2T and expressed in bacteria as GST fusion proteins as previously
described.23 (link) Kinase assays were performed
in a reaction mixture containing 0.5 μg of GST-SRPK1, 1.5 μg
of GST-LBRNt(62–92) as a substrate, 25 μM ATP, 1 μCi
of [γ-32P]ATP, 12 mM Hepes pH 7.5, 10 mM MgCl2, and the appropriate amount of the inhibitor in a final volume
of 25 μL. Reactions were carried out for 30 min at 30 °C.
The final concentration of dimethyl sulfoxide (DMSO) was adjusted
to 4% irrespectively of the inhibitor concentration.
Human recombinant
casein kinase 2 (CK2) was purchased from New England Biolabs. The
CK2 kinase assay was performed in a reaction mixture containing 100
units of CK2, 1.5 μg of dephosphorylated casein (Sigma), 25
μM ATP, 1 μCi of [γ-32P]ATP, 50 mM Tris-HCl
pH 7.5, 10 mM MgCl2, and the appropriate amount of the
inhibitor in a final volume of 25 μL. Reactions were carried
out for 30 min at 30 °C. The final concentration of dimethyl
sulfoxide (DMSO) was adjusted to 4% irrespectively of the inhibitor
concentration. Phosphoproteins were detected by autoradiography using
Super RX (Fuji medical X-ray film), and incorporation of radioactivity
was measured by excising the radioactive bands from the SDS-PAGE gel
and scintillation counting.
and a fragment of the N-terminal domain of turkey Lamin B Receptor
(LBR) comprising amino acids 62–92 (LBRNt(62-92)) were subcloned
into pGEX-2T and expressed in bacteria as GST fusion proteins as previously
described.23 (link) Kinase assays were performed
in a reaction mixture containing 0.5 μg of GST-SRPK1, 1.5 μg
of GST-LBRNt(62–92) as a substrate, 25 μM ATP, 1 μCi
of [γ-32P]ATP, 12 mM Hepes pH 7.5, 10 mM MgCl2, and the appropriate amount of the inhibitor in a final volume
of 25 μL. Reactions were carried out for 30 min at 30 °C.
The final concentration of dimethyl sulfoxide (DMSO) was adjusted
to 4% irrespectively of the inhibitor concentration.
Human recombinant
casein kinase 2 (CK2) was purchased from New England Biolabs. The
CK2 kinase assay was performed in a reaction mixture containing 100
units of CK2, 1.5 μg of dephosphorylated casein (Sigma), 25
μM ATP, 1 μCi of [γ-32P]ATP, 50 mM Tris-HCl
pH 7.5, 10 mM MgCl2, and the appropriate amount of the
inhibitor in a final volume of 25 μL. Reactions were carried
out for 30 min at 30 °C. The final concentration of dimethyl
sulfoxide (DMSO) was adjusted to 4% irrespectively of the inhibitor
concentration. Phosphoproteins were detected by autoradiography using
Super RX (Fuji medical X-ray film), and incorporation of radioactivity
was measured by excising the radioactive bands from the SDS-PAGE gel
and scintillation counting.
4
Polo Kinase Phosphorylation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full-length Polo kinase cDNA was cloned into a Gateway baculovirus expression construct (Invitrogen) with a hexahistidine (6×His) tag. Sf9 cells were infected and then harvested on a nickel column at a concentration of 0.5 mg/ml. GST-tagged Nuf (Rothwell et al., 1999 (link)) was purified using glutathione–Sepharose beads to a concentration of 1 mg/ml. Dephosphorylated casein (Sigma-Aldrich) was dissolved in water to 1 mg/ml. Kinase reactions were assembled using 5 μg of substrate (casein or GST-Nuf), 0.05 mM ATP, 0.05 μg of Polo-6×His, 5 μCi of ATP32, and kinase buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 75 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM ethylene glycol tetraacetic acid). Extracts from Sf9 cells infected with empty virus were used as control kinases at 0.5 mg/ml. The 25-μl reactions were carried out at 30°C for 20 min and then boiled in 2× sample buffer and run on SDS–PAGE. Nonradiolabeled Polo phosphorylated bands were gel extracted and used for tandem mass spectrometry. Mass spectrometry was performed by the Bio-Organic Biomedical Mass Spectrometry Resource at the University of California, San Francisco, using standard protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!