GDE / AGL | Glycogen debranching enzyme
AS09 454 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Human, Mouse, Rabbit, Rat
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(1) rat liver homogenate (50 µg of total protein), (2) GDE IP from 500 µg rat liver homogenate (3) GDE IP from 10 µg purified rat liver glycogen (see Parker et al, BBRC 2007) 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 GDE antibodies (diluted 1/2000) 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 withchemiluminescent detection reagent. Images were detected using the Fuji LAS-3000 system.
To obtain GDE-bound immunoprecipitates, 5 µg GDE antibody was incubated with 500 µg rat liver homogenate or 10 ug purified rat liver glycogen 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. 100µ l, 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.
This antibody can detect GDE in crude tissue homogenate in both, liver and muscle tissue and is able to immunoprecipiate GDE.
Antibody will work as an excellent marker for glycogen. In tissues with no glycogen extression there will be no signal using this antibody.
Synonymes:Glycogen debrancher, 4-alpha-glucanotransferase (EC=188.8.131.52), Oligo-1,4-1,4-glucantransferase, amylo-alpha-1,6-glucosidase, amylo-1,6-glucosidase (EC=184.108.40.206), Dextrin 6-alpha-D-glucosidase,amylo-1, 6-glucosidase, 4-alpha-glucanotransferase
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