GP | Glycogen phosphorylase
AS09 455 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Chicken, Human, Mouse, Rabbit, Rat, T.vaginalis G3
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Atlantic Salmon, Bovine, Dog, Drosophila melanogaster, Hen, Horse, Pig, Zebrafish, Xenopus laevis
(1) rat liver homogenate (50 µg of total protein), (2) 10 ug purified rat liver glycogen (see Parker et al, BBRC 2007), (3) Human skeletal muscle homogenate (50 µg of total protein), (4) GP IP from 500 ug Human skeletal muscle homogenate were resuspended into SDS PAGE sample buffer, boiled, electrophoresed on a pre-cast 4-15% gradient gel (Invitrogen, CA, USA) and transferred to PVDF membrane. Following blocking in 5% milk / PBST, membranes were probed with GP antibodies (diluted 1/1000) for 1h at room temperature with rocking, followed by washing in 3 times for 5 min in PBS-T at room temperature with agitation. Blots were incubated in secondary antibody (Protein-G coupled to horse radish peroxidase conjugated – Bio-Rad) diluted 1:3000 for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescent detection reagent. Images were detected using the Fuji LAS-3000 system.
To obtain GP-bound immunoprecipitates, 5 ug GP antibody was incubated with 500 ug human skeletal muscle homogenate together with 1 ml IP wash buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EGTA, 10 mM NaPPi and 100 mM NaF) and incubated at 4°C with mixing for 90 minutes. 100 ul, 20% Protein-A Sepharose was then added for a further 30 minutes at 4°C with mixing. Immunoprecipitates were collected by centrifugation at 7,000 rpm for 1 minutes and washed three times with IP wash buffer followed by resuspension into SDS PAGE sample buffer, boiling and electrophoresis as above.
The GP antibody did not immunoprecipitate glycogen phosphorylase from rat liver.
Courtesy of Dr. Melissa Stuart, Microbiology/Immunology Kirksville College of Osteopathic Medicine, USA
This antibody can detect GP in crude tissue homogenate in both, liver and muscle tissue and is able to immunoprecipiate GP.
Glycogen phosphorylase is a good marker for determining the degree of postmortem protein denaturation in muscle. When glycogen phosphorylase denatures it shifts from the sarcoplasmic fraction of the muscle to the myofibrillar fraction of the muscle. This shift is detectable using SDS-PAGE and western blotting techniques. The antibody is being used to detect glycogen phosphorylase levels in both sarcoplasmic and myofibrillar protein extracts from chicken muscle.
Glycogen phosphorylase (EC 18.104.22.168) is an enzyme which is catalyzing the rate limiting step in the degradation of glycogen in animals by releasing glucose-1-phosphate from the terminal alpha1,4-glycosidic bond.
Bowker and Zhuang (2015). Relationship between water-holding capacity and protein denaturation in broiler breast meat. Poult Sci. 2015 May 25. pii: pev120.
Zhu et al. (2011). High post-mortem temperature combined with rapid glycolysis induces phosphorylase denaturation and produces pale and exudative characteristics in broiler Pectoralis major muscles. Meat Science