Western blot analysis of Baculovirus expressed PKCβ and PKCβ Ser660/Ala mutant, using Phospho-PKC (pan) (βII Ser660) Antibody (upper) or control PKCβ antibody (lower).
Western blot analysis of extracts from TPA, Go6983 and/or Bisindolylmaleimide treated 293 cells, using Phospho-PKC (pan) (βII Ser660) Antibody.
Western blot analysis of Baculovirus expressed PKC isoforms, using Phospho-PKC (pan) (βII Ser660) Antibody.
|REACTIVITY||H M R Mk|
|MW (kDa)||78, 80, 82, 85|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-PKC (pan) (βII Ser660) Antibody detects endogenous levels of PKC α, β I, β II, δ, ε, η and θ isoforms only when phosphorylated at a carboxy-terminal residue homologous to serine 660 of PKC β II. This antibody does not detect PKC phosphorylated at other sites.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser660 of human PKC β II. Antibodies are purified by protein A and peptide affinity chromatography.
Activation of protein kinase C (PKC) is one of the earliest events in a cascade that controls a variety of cellular responses, including secretion, gene expression, proliferation, and muscle contraction (1,2). PKC isoforms belong to three groups based on calcium dependency and activators. Classical PKCs are calcium-dependent via their C2 domains and are activated by phosphatidylserine (PS), diacylglycerol (DAG), and phorbol esters (TPA, PMA) through their cysteine-rich C1 domains. Both novel and atypical PKCs are calcium-independent, but only novel PKCs are activated by PS, DAG, and phorbol esters (3-5). Members of these three PKC groups contain a pseudo-substrate or autoinhibitory domain that binds to substrate-binding sites in the catalytic domain to prevent activation in the absence of cofactors or activators. Control of PKC activity is regulated through three distinct phosphorylation events. Phosphorylation occurs in vivo at Thr500 in the activation loop, at Thr641 through autophosphorylation, and at the carboxy-terminal hydrophobic site Ser660 (2). Atypical PKC isoforms lack hydrophobic region phosphorylation, which correlates with the presence of glutamic acid rather than the serine or threonine residues found in more typical PKC isoforms. The enzyme PDK1 or a close relative is responsible for PKC activation. A recent addition to the PKC superfamily is PKCμ (PKD), which is regulated by DAG and TPA through its C1 domain. PKD is distinguished by the presence of a PH domain and by its unique substrate recognition and Golgi localization (6). PKC-related kinases (PRK) lack the C1 domain and do not respond to DAG or phorbol esters. Phosphatidylinositol lipids activate PRKs, and small Rho-family GTPases bind to the homology region 1 (HR1) to regulate PRK kinase activity (7).
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