PKCγ Antibody (Rabbit mAb) [F18C13]

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
WB1:1000 IP1:30 IHC1:100 IF1:200

Application
WB, IP, IHC, IF

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
78 kDa 80 kDa,36 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
PKCγ Antibody (Rabbit mAb) [F18C13] detects endogenous levels of total PKCγ protein.

Clone
F18C13

Synonym(s)
PKCG, PRKCG, Protein kinase C gamma type, PKC-gamma

Background
PKCγ belongs to the conventional subgroup of the protein kinase C family, alongside PKCα and PKCβ, and is distinguished from other PKC isozymes by expression restricted almost entirely to the central nervous system, with particularly high abundance in cerebellar Purkinje cells. Like other conventional PKC isoforms, PKCγ is held in an autoinhibited, catalytically inactive state by a pseudosubstrate sequence that occupies the substrate-binding groove of the catalytic domain, and this autoinhibition is relieved when calcium and diacylglycerol engage the C2 and tandem C1A/C1B regulatory domains, respectively, releasing the pseudosubstrate and allowing the kinase domain to phosphorylate downstream target proteins. The great majority of pathogenic PRKCG variants causing spinocerebellar ataxia type 14 cluster within the C1A and C1B regulatory domains, with additional variants found in the C2 domain and, less frequently, in the catalytic domain itself, and biochemical characterization of C1A domain variants shows that they produce increased basal kinase activity while simultaneously becoming unresponsive to normal agonist-driven stimulation and relatively resistant to receptor-mediated downregulation, a pattern consistent with a leaky, partially deautoinhibited kinase rather than a simply hyperactive one. Functional testing of individual SCA14-associated mutations using Purkinje cell dendritic morphology as a live-cell readout of PKCγ biological activity shows that catalytic-domain mutations reliably produce increased biological activity in Purkinje cells, whereas mutations located within the regulatory C1 or C2 domains do not show this increased activity by the same dendritic-growth assay, despite several of these regulatory-domain mutations showing elevated enzymatic activity in isolated biochemical assays; this discrepancy indicates that regulatory-domain and catalytic-domain SCA14 mutations disrupt Purkinje cell function through mechanistically distinct routes rather than through a single, shared gain-of-function pathway. Independently of catalytic output, both wild-type and mutant PKCγ can form amyloid-like fibrils and aggregates in overexpression and reconstituted systems, and such aggregates are additionally detected in induced pluripotent stem cells derived from SCA14 patients and in primary Purkinje cell cultures, positioning PKCγ aggregation as a separate, mutation-associated pathological process running in parallel to altered catalytic regulation. PKCγ additionally functions in developmental synapse elimination, specifically pruning climbing fiber synapses from developing Purkinje cells, and disruption of this normal PKCγ-dependent pruning and dendritic development process by SCA14 mutations is directly implicated in the progressive cerebellar ataxia and Purkinje cell dysfunction characteristic of the disease.

Background

PKCγ belongs to the conventional subgroup of the protein kinase C family, alongside PKCα and PKCβ, and is distinguished from other PKC isozymes by expression restricted almost entirely to the central nervous system, with particularly high abundance in cerebellar Purkinje cells. Like other conventional PKC isoforms, PKCγ is held in an autoinhibited, catalytically inactive state by a pseudosubstrate sequence that occupies the substrate-binding groove of the catalytic domain, and this autoinhibition is relieved when calcium and diacylglycerol engage the C2 and tandem C1A/C1B regulatory domains, respectively, releasing the pseudosubstrate and allowing the kinase domain to phosphorylate downstream target proteins. The great majority of pathogenic PRKCG variants causing spinocerebellar ataxia type 14 cluster within the C1A and C1B regulatory domains, with additional variants found in the C2 domain and, less frequently, in the catalytic domain itself, and biochemical characterization of C1A domain variants shows that they produce increased basal kinase activity while simultaneously becoming unresponsive to normal agonist-driven stimulation and relatively resistant to receptor-mediated downregulation, a pattern consistent with a leaky, partially deautoinhibited kinase rather than a simply hyperactive one. Functional testing of individual SCA14-associated mutations using Purkinje cell dendritic morphology as a live-cell readout of PKCγ biological activity shows that catalytic-domain mutations reliably produce increased biological activity in Purkinje cells, whereas mutations located within the regulatory C1 or C2 domains do not show this increased activity by the same dendritic-growth assay, despite several of these regulatory-domain mutations showing elevated enzymatic activity in isolated biochemical assays; this discrepancy indicates that regulatory-domain and catalytic-domain SCA14 mutations disrupt Purkinje cell function through mechanistically distinct routes rather than through a single, shared gain-of-function pathway. Independently of catalytic output, both wild-type and mutant PKCγ can form amyloid-like fibrils and aggregates in overexpression and reconstituted systems, and such aggregates are additionally detected in induced pluripotent stem cells derived from SCA14 patients and in primary Purkinje cell cultures, positioning PKCγ aggregation as a separate, mutation-associated pathological process running in parallel to altered catalytic regulation. PKCγ additionally functions in developmental synapse elimination, specifically pruning climbing fiber synapses from developing Purkinje cells, and disruption of this normal PKCγ-dependent pruning and dendritic development process by SCA14 mutations is directly implicated in the progressive cerebellar ataxia and Purkinje cell dysfunction characteristic of the disease.

References
https://pubmed.ncbi.nlm.nih.gov/28738819/ https://pubmed.ncbi.nlm.nih.gov/40100287/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%