Anti-PKC gamma (phospho T655) antibody (ab5796)
Key features and details
- Rabbit polyclonal to PKC gamma (phospho T655)
- Suitable for: WB
- Reacts with: Human
- Isotype: IgG
Overview
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Product name
Anti-PKC gamma (phospho T655) antibody
See all PKC gamma primary antibodies -
Description
Rabbit polyclonal to PKC gamma (phospho T655) -
Host species
Rabbit -
Specificity
PKC alpha (69%) and beta 2 (62%) may cross-react in cells expressing high levels of these proteins. -
Tested Applications & Species
See all applications and species dataApplication Species WB Human
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Immunogen
Synthetic phosphopeptide derived from a region of human PKC gamma that contains threonine 655.
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Positive control
- Hela cells treated with PMA, a phorbol ester.
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General notes
PKC gamma is an 80 kDa member of the conventional group (cPKCs: sensitive to calcium, diacylglycerol and phorbol esters) of the PKC family of serine/threonine family kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival and transcriptional regulation. PKC gamma plays a key role in neuronal signal transduction and is translocated from the nucleus to the cytoplasm upon activation by phorbol ester, where in epithelial cells it has been implicated in regulating intracellular communication. The activation loop threonine (threonine 514 in PKC gamma) of conventional PKCs is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1), which is necessary for their autophosphorylation, a critical step in the generation of a catalytically mature enzyme. Threonine 655 is an autophosphorylation site in the carboxy-terminus of PKC gamma.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles. -
Storage buffer
pH: 7.30
Preservative: 0.05% Sodium azide
Constituents: PBS, 0.1% BSA -
Concentration information loading... -
Purity
Immunogen affinity purified -
Purification notes
The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PKC gamma. The final product is generated by affinity chromatography using a PKC gamma-derived peptide that is phosphorylated at threonine 655. -
Primary antibody notes
PKC gamma is an 80 kDa member of the conventional group (cPKCs: sensitive to calcium, diacylglycerol and phorbol esters) of the PKC family of serine/threonine family kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival and transcriptional regulation. PKC gamma plays a key role in neuronal signal transduction and is translocated from the nucleus to the cytoplasm upon activation by phorbol ester, where in epithelial cells it has been implicated in regulating intracellular communication. The activation loop threonine (threonine 514 in PKC gamma) of conventional PKCs is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1), which is necessary for their autophosphorylation, a critical step in the generation of a catalytically mature enzyme. Threonine 655 is an autophosphorylation site in the carboxy-terminus of PKC gamma. -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Images
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Western blot - Anti-PKC gamma (phospho T655) antibody (ab5796)
Peptide Competition and Phosphatase treatment: Lysates prepared from HeLa cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-11) or treated with lambda (ë) phosphatase (12), blocked with a 3% low-fat milk-TBST buffer overnight at 4°C, and incubated with 0.50
µ g/mL ab5796 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: no peptide (1, 11, 12), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-10). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStarTM method. The data show that the peptide corresponding to PKC gamma [pT655] blocks the antibody signal while the peptides corresponding to PKC isoforms beta 1 [pT642], delta [pS645], epsilon [pT710] and eta [pT655] do not completely block the antibody signal, thereby demonstrating the specificity of the antibody. However, peptides corresponding to PKC isoforms alpha [pT638] and beta 2 [pT641] do block the antibody signal, indicating that the ab5796 antibody may cross-react with their respective sites. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.
Peptide Competition and Phosphatase treatment: Lysates prepared from HeLa cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-11) or treated with lambda (ë) phosphatase (12), blocked with a 3% low-fat milk-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5796 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: no peptide (1, 11, 12), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-10). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStarTM method. The data show that the peptide corresponding to PKC gamma [pT655] blocks the antibody signal while the peptides corresponding to PKC isoforms beta 1 [pT642], delta [pS645], epsilon [pT710] and eta [pT655] do not completely block the antibody signal, thereby demonstrating the specificity of the antibody. However, peptides corresponding to PKC isoforms alpha [pT638] and beta 2 [pT641] do block the antibody signal, indicating that the ab5796 antibody may cross-react with their respective sites. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.
