Invitrogen

Phospho-PKC gamma (Thr514) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Peptide array to ensure that the antibody binds to the antigen stated.

2 References

View all (23) PKC gamma antibodies

Datasheet

Protocols

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Datasheet

Protocols

Questions & Answers

Tech Support

Product Image Gallery

Phospho-PKC gamma (Thr514) Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of Phospho PKC GAMMA (pT514) showing staining in the cytoplasm of paraffin-embedded human skin tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho PKC GAMMA (pT514) Rabbit Polyclonal Antibody (Product # 44-956) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Phospho-PKC gamma (Thr514) Antibody in Western Blot (WB) — 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 (1-10) or treated with lambda phosphatase (11), blocked with a 3% low-fat milk-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL PKCgamma (pT514) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 10, 11), 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 (5-9). After washing, membranes were incubated with goat F (ab')2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that the peptide corresponding to PKCgamma (pS514) blocks the antibody signal and that the peptides corresponding to PKC isoforms alpha (pT497), betaI&II (pT500), delta (pT507), epsilon (pT710) and theta (pT538) did not block the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Phospho-PKC gamma (Thr514) Antibody — Antibody specificity is demonstrated by Peptide Array. Western blotting of Phospho-PKC gamma (Thr514) using anti-Phospho-PKC gamma (Thr514) Polyclonal Antibody (Product # 44-956) shows loss of signal with the specific peptide and not with other relevant peptides. {ARRAY}

Product Details

44-956

Applications	Tested Dilution	Publications
Western Blot (WB)	1:1,000	View 2 publications 2 publications
Immunohistochemistry (Paraffin) (IHC (P))	1:10-1:100	-

Product Specifications
Species Reactivity	Human, Mouse
Published species	Hamster
Host/Isotype	Rabbit / IgG
Class	Polyclonal
Type	Antibody
Immunogen	The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human PKC-gamma that contains threonine 514 View immunogen
Conjugate	Unconjugated
Form	Liquid
Purification	Antigen affinity chromatography
Storage buffer	Dulbecco's PBS, pH 7.3, with 1mg/mL BSA
Contains	0.05% sodium azide
Storage conditions	-20°C
RRID	AB_2533806

Target Information

Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play distinct roles in cells. PKC gamma is one of the PKC family members. This protein kinase is expressed solely in the brain and spinal cord and its localization is restricted to neurons. It has been demonstrated that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require this kinase. Knockout studies in mice also suggest that this kinase may be involved in neuropathic pain development. Defects in this protein have been associated with neurodegenerative disorder spinocerebellar ataxia-14 (SCA14).

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Product References

Mutant protein kinase Cgamma found in spinocerebellar ataxia type 14 is susceptible to aggregation and causes cell death.

The Journal of biological chemistry

Seki T,Adachi N,Ono Y,Mochizuki H,Hiramoto K,Amano T,Matsubayashi H,Matsumoto M,Kawakami H,Saito N,Sakai N

44-956 was used in Western Blotting to indicate that spinocerebellar ataxia type 14 (SCA14) mutations make protein kinase Cgamma (gammaPKC) form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.