GCLC Antibody (Rabbit mAb) [D14E17]

Application: Reactivity: Mouse, Rat, Human

Usage Information

Dilution
WB1:500-1:1000 IP1:40 IF1:100 FCM1:60

Application
WB, IP, IF, FCM

Reactivity
Mouse, Rat, Human

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
73 kDa 73 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.

Positive Control	Human lung tissue; Human fetal liver tissue; Rat liver tissue; Mouse liver tissue; Mouse spleen tissue; HepG2 cells; MCF7 cells; PC-12 cells; A549 cells; C6 cells; RAW 264.7 cells
Negative Control

Datasheet & SDS

Biological Description

Specificity
GCLC Antibody (Rabbit mAb) [D14E17] detects endogenous levels of total GCLC protein.

Clone
D14E17

Synonym(s)
GLCL, GLCLC, GCLC, Glutamate--cysteine ligase catalytic subunit, GCS heavy chain, Gamma-ECS, Gamma-glutamylcysteine synthetase

Background
GCLC is the catalytic subunit of glutamate-cysteine ligase, the enzyme that catalyzes the ATP-dependent condensation of L-glutamate and L-cysteine into gamma-glutamylcysteine, constituting the first and rate-limiting step in de novo glutathione biosynthesis. GCLC carries the enzyme's active site and retains catalytic activity when expressed alone, but its function is directly modulated by association with a separate regulatory subunit, GCLM: heterodimerization of GCLC with GCLM lowers the Michaelis constant for glutamate and raises the inhibition constant for glutathione, together producing a two- to five-fold increase in catalytic efficiency compared with GCLC acting alone, and this heterodimer assembly represents a defined allosteric activation mechanism operating independently of transcriptional control. GCLC activity is additionally subject to direct feedback inhibition by glutathione, the end product of the pathway it initiates, establishing a self-limiting biochemical loop in which glutathione accumulation restrains further glutamate-cysteine ligase activity without requiring changes in gene expression. Transcriptional control of GCLC is mediated through antioxidant response elements located in its promoter region, and chromatin immunoprecipitation confirms that the transcription factor Nrf2 associates directly with a functional antioxidant response element in the GCLC promoter, with Nrf2 co-expression driving several-fold activation of GCLC promoter-reporter constructs; this Nrf2-ARE regulatory axis couples GCLC transcription to the same oxidative-stress-sensing pathway that governs the broader Nrf2-dependent antioxidant gene program. GCLC and its regulatory partner GCLM are not always co-regulated, since GCL subunit genes show cell-type-specific differences in both basal and inducible expression, with some inducing agents preferentially upregulating GCLM over GCLC or failing to induce either subunit depending on the specific cell line examined, indicating that GCLC transcriptional control operates through partially independent, cell-context-dependent regulatory circuits rather than a single uniform mechanism across tissues. Glutathione is the principal intracellular antioxidant buffer protecting cells against oxidative and electrophilic stress; GCLC-driven glutathione synthesis is directly implicated in cellular defense against chronic hyperglycemic and oxidative insults, and disruption of Nrf2-dependent GCLC induction is linked to impaired redox homeostasis relevant to diabetic vascular complications and to neurodegenerative and other oxidative-stress-associated disease states.

Background

GCLC is the catalytic subunit of glutamate-cysteine ligase, the enzyme that catalyzes the ATP-dependent condensation of L-glutamate and L-cysteine into gamma-glutamylcysteine, constituting the first and rate-limiting step in de novo glutathione biosynthesis. GCLC carries the enzyme's active site and retains catalytic activity when expressed alone, but its function is directly modulated by association with a separate regulatory subunit, GCLM: heterodimerization of GCLC with GCLM lowers the Michaelis constant for glutamate and raises the inhibition constant for glutathione, together producing a two- to five-fold increase in catalytic efficiency compared with GCLC acting alone, and this heterodimer assembly represents a defined allosteric activation mechanism operating independently of transcriptional control. GCLC activity is additionally subject to direct feedback inhibition by glutathione, the end product of the pathway it initiates, establishing a self-limiting biochemical loop in which glutathione accumulation restrains further glutamate-cysteine ligase activity without requiring changes in gene expression. Transcriptional control of GCLC is mediated through antioxidant response elements located in its promoter region, and chromatin immunoprecipitation confirms that the transcription factor Nrf2 associates directly with a functional antioxidant response element in the GCLC promoter, with Nrf2 co-expression driving several-fold activation of GCLC promoter-reporter constructs; this Nrf2-ARE regulatory axis couples GCLC transcription to the same oxidative-stress-sensing pathway that governs the broader Nrf2-dependent antioxidant gene program. GCLC and its regulatory partner GCLM are not always co-regulated, since GCL subunit genes show cell-type-specific differences in both basal and inducible expression, with some inducing agents preferentially upregulating GCLM over GCLC or failing to induce either subunit depending on the specific cell line examined, indicating that GCLC transcriptional control operates through partially independent, cell-context-dependent regulatory circuits rather than a single uniform mechanism across tissues. Glutathione is the principal intracellular antioxidant buffer protecting cells against oxidative and electrophilic stress; GCLC-driven glutathione synthesis is directly implicated in cellular defense against chronic hyperglycemic and oxidative insults, and disruption of Nrf2-dependent GCLC induction is linked to impaired redox homeostasis relevant to diabetic vascular complications and to neurodegenerative and other oxidative-stress-associated disease states.

References
https://pubmed.ncbi.nlm.nih.gov/19540342/ https://pubmed.ncbi.nlm.nih.gov/18812186/

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%