Immunofluorescence analysis of Gamma-Glutamyl cysteine Synthetase/Glutamate-cysteine was performed using70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with GCLC Rabbit Polyclonal Antibody (PA516581) at 2µg/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytosolic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Bovine, Human, Mouse, Non-human primate, Rat|
|Published species reactivity||Rat, Human, Mouse, Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||A synthetic peptide derived from corresponding to aa 295-313 of rat GCLC (heavy subunit of GCS)|
|Storage buffer||PBS, pH 7.4, with 0.2% BSA|
|Contains||0.09% sodium azide|
|Storage Conditions||4° C|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunoprecipitation (IP)||10 µg/ml|
|Western Blot (WB)||2.5-5.0 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 11 publications below|
PA5-16581 targets Gamma-Glutamyl cysteine Synthetase/Glutamate-cysteine Ligase in IP and WB applications and shows reactivity with Bovine, Human, mouse, Non-human primate, and Rat samples.
The PA5-16581 immunogen is a synthetic peptide derived from corresponding to aa 295-313 of rat GCLC (heavy subunit of GCS).
Glutathione (GSH) plays an important role in detoxification of oxidants and toxins from the cells. In most cells GSH is synthesized de novo in two steps catalysed by Gamma Glutamylcysteine Synthetase (GCS)/Glutamate-cystein Ligase and GSH synthetase. GCS catalyses the first and rate limiting step and is important in GSH homeostasis. It is composed of two subunits: heavy and light. These subunits are coded by different genes which are controlle by different mechanisms. The heavy subunit carries the catalytic activity and can be inhibited through feedback mechanism by GSH. Binding of light subunit to heavy subunit reduces the Michaelis-Menten constant for glutamate to a physiological concentration of 1.2mM and increases the inhibitory constant for GSH.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine.
PA5-16581 was used in western blot to study the effects of betaine administration on rat kidney sulfur compound metabolomics
|Kim YC,Kwon DY,Kim JH||Amino acids (46:963)||2014|
Does hepatic oxidative stress enhance activation of nuclear factor-E2-related factor in patients with nonalcoholic steatohepatitis?
PA5-16581 was used in western blot to study the effects of oxidative stress on the expression of Nrf2 in the livers of patients with non-alcoholic steatohepatitis
|Takahashi Y,Kobayashi Y,Kawata K,Kawamura K,Sumiyoshi S,Noritake H,Watanabe S,Chida T,Souda K,Sakaguchi T,Nakamura H,Suda T||Antioxidants and redox signaling (20:538)||2014|
Impaired transcriptional activity of Nrf2 in age-related myocardial oxidative stress is reversible by moderate exercise training.
PA5-16581 was used in western blot to study the ability of moderate exercise to reverse the age-related decline in cardiac Nrf2 transcriptional activity and its associated antioxidants
|Gounder SS,Kannan S,Devadoss D,Miller CJ,Whitehead KJ,Whitehead KS,Odelberg SJ,Firpo MA,Paine R,Hoidal JR,Abel ED,Rajasekaran NS||PloS one (7:null)||2012|
Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis).
PA5-16581 was used in western blot to study the ability of a green perilla leaf extract to activate the Nrf2-ARE pathway
|Izumi Y,Matsumura A,Wakita S,Akagi K,Fukuda H,Kume T,Irie K,Takada-Takatori Y,Sugimoto H,Hashimoto T,Akaike A||Free radical biology and medicine (53:669)||2012|
Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle.
PA5-16581 was used in immunohistochemistry and western blot to study the potential role of Nrf2 signaling in age-related ROS accumulation and skeletal muscle myopathy
|Miller CJ,Gounder SS,Kannan S,Goutam K,Muthusamy VR,Firpo MA,Symons JD,Paine R,Hoidal JR,Rajasekaran NS||Biochimica et biophysica acta (1822:1038)||2012|
Cross-regulations among NRFs and KEAP1 and effects of their silencing on arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.
PA5-16581 was used in western blot to study the regulatory interactions of NRF2, NRF1, and KEAP1 and their role in the anti-oxidant responses of human keratinocytes to arsenic toxicity
|Zhao R,Hou Y,Zhang Q,Woods CG,Xue P,Fu J,Yarborough K,Guan D,Andersen ME,Pi J||Environmental health perspectives (120:583)||2012|
Microtubules as a critical target for arsenic toxicity in lung cells in vitro and in vivo.
PA5-16581 was used in western blot to study the role of microtubules as targets of lung cell arsenic toxicity and the mechanisms involved
|Zhao Y,Toselli P,Li W||International journal of environmental research and public health (9:474)||2012|
Increased glutathione synthesis following Nrf2 activation by vanadyl sulfate in human chang liver cells.
PA5-16581 was used in western blot to study the effect of vanadyl sulfate on nrf2 activation and glutathione synthesis in human chang liver cells
|Kim AD,Zhang R,Kang KA,You HJ,Hyun JW||International journal of molecular sciences (12:8878)||2012|
Acute exercise stress activates Nrf2/ARE signaling and promotes antioxidant mechanisms in the myocardium.
PA5-16581 was used in western blot to study the effect of acute exercise stress on Nrf2/ARE signaling and antioxidant mechanisms in the myocardium
|Muthusamy VR,Kannan S,Sadhaasivam K,Gounder SS,Davidson CJ,Boeheme C,Hoidal JR,Wang L,Rajasekaran NS||Free radical biology and medicine (52:366)||2012|
|Not Applicable||Not Cited||
Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate-cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling.
PA5-16581 was used in western blot to study the potential role of carbon monoxide produced by heme oxygenase-1 in the adaptive survival response to peroxynitrite-induced PC12 cell death
|Li MH,Jang JH,Na HK,Cha YN,Surh YJ||The Journal of biological chemistry (282:28577)||2007|
Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.
PA5-16581 was used in western blot to examine the role of antioxidant enzymes in rat retinal ischemia-reperfusion injury and recovery
|Agardh CD,Gustavsson C,Hagert P,Nilsson M,Agardh E||Metabolism: clinical and experimental (55:892)||2006|