Application
Extracts of HeLa cells untreated (1-7) or treated with 1µM PMA for 30 minutes (8-14) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either left untreated (1-13) or treated with lambda phosphatase (14), blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the PKCgamma [pT674] antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: the phosphopeptide corresponding to the immunogen from other PKC isoforms (1-9), the phosphopeptide immunogen (10), the non-phosphopeptide corresponding to the phosphopeptide immunogen (11), a generic phosphothreonine-containing peptide (12) or no peptide (13, 14). After washing, the membrane was incubated with goat F(ab)2 anti-rabbit IgG HRP-conjugate and signals were detected. The data show that the phosphopeptide corresponding to PKCgamma [pT674] blocks the antibody signal. The peptides corresponding to PKCalpha [pT657], betaI [pS661] and PKCeta [pS674] also block the antibody signal, suggesting cross-reactivity of the PKCalpha [pT638] antibody with these sites. The antibody signal was not blocked by the peptides corresponding to PKC isoforms betaII [pT660], gamma [pT514], gamma [pT655], delta [pS664], theta [pS695] and zeta [pT560], demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.| Product Name | PKC gamma (phospho Thr674) antibody |
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| Antibody Type | Primary Antibodies |
| Product description | Rabbit polyclonal to PKC gamma ( phospho T674 )Protein Kinase C gamma (PKC gamma) is an 80 kDa member of the conventional grou(cPKCs: sensitive to calcium, diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine 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 in regulating intercellular 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 its autophosphorylation on threonine 655 in the turn loop, and threonine 674 in the hydrophobic loop of the carboxy terminus, a critical step in generating a catalytically mature enzyme. The phosphorylation of the hydrophobic loop in the carboxyl terminus of PKCs is believed to be a key determinant in regulating PKC interaction with PDK1. |
| Immunogen | Synthetic phosphopeptide derived from a region of human PKC gamma that contains threonine 674. |
| Modification | p-Thr674 |
| Clonality | Polyclonal |
|---|---|
| Isotype | IgG |
| Host Species | Rabbit |
| Tested Applications | WB |
| WB:1/1000. Detects a band of approximately 80 kDa. Optimal dilutions/concentrations should be determined by the end user. | |
| Species Reactivity | HumanMouseRat |
| Concentration | 1mg/ml |
| Purification | Affinity purified |
| Gene Synonyms | MGC57564, PKC-gamma, PKCC, PKCG, SCA14, PKC gamma, PKCgamma, PKC gamma (phospho T674), PKC gamma (phospho Thr674), PKC gamma (pT674), PKC gamma (pThr674), PKC gamma phospho T674, PKC gamma phospho Thr674, Phospho PKC gamma (T674), Phospho PKC gamma (Thr67 |
|---|---|
| Target | PKC gamma |
| Tissue Specificity | PKC alpha (69%) may cross-react in cells expressing a high level of this protein. The peptide competition data (see Figure) suggest that this antibody will cross-react with PKC alpha [pS657] (69% homologous), beta 1 [pS661] (54%) and PKC eta [pS674] (38%). |
Application
Extracts of HeLa cells untreated (1-7) or treated with 1µM PMA for 30 minutes (8-14) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either left untreated (1-13) or treated with lambda phosphatase (14), blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the PKCgamma [pT674] antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: the phosphopeptide corresponding to the immunogen from other PKC isoforms (1-9), the phosphopeptide immunogen (10), the non-phosphopeptide corresponding to the phosphopeptide immunogen (11), a generic phosphothreonine-containing peptide (12) or no peptide (13, 14). After washing, the membrane was incubated with goat F(ab)2 anti-rabbit IgG HRP-conjugate and signals were detected. The data show that the phosphopeptide corresponding to PKCgamma [pT674] blocks the antibody signal. The peptides corresponding to PKCalpha [pT657], betaI [pS661] and PKCeta [pS674] also block the antibody signal, suggesting cross-reactivity of the PKCalpha [pT638] antibody with these sites. The antibody signal was not blocked by the peptides corresponding to PKC isoforms betaII [pT660], gamma [pT514], gamma [pT655], delta [pS664], theta [pS695] and zeta [pT560], demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.| Application Notes | WB:1/1000. Detects a band of approximately 80 kDa. Optimal dilutions/concentrations should be determined by the end user. |
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| Form | Liquid |
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| Storage Instructions | Upon receipt - Keep as concentrated solution. Aliquot and store at -20ºC or below. Avoid freeze-thaw cycles. |
| Storage Buffer | Preservative: 0.05% Sodium Azide; Constituents: PBS, 50% glycerol, 1mg/ml BSA. pH 7.3 |
