Tissues were homogenised in lysis buffer (20 mM Tris, 1 mM EDTA and 0.25 mM sucrose, pH 7.4) and ultracentrifuged to obtain the membrane fractions. The amount of protein in the homogenate was quantified using the Bio-Rad Protein Assay, based on the Bradford method. Equivalent amounts of protein were resolved on an 8–12% SDS-PAGE and transferred onto a PVDF membrane (Immobilon-P transfer membrane, Millipore). Glycogen synthase was detected using the Abcam (EP817Y, ab40810) rabbit monoclonal anti-gys1 primary antibody. GLUT-4 was detected using the Abcam (mAbcam65267) anti-GLUT-4 primary antibody. Hexokinase II was detected using the Abcam (ab76959) mouse monoclonal Anti-Hexokinase II antibody. Glucose-6-phosphate isomerase was detected using the Abcam [1B7D7] (ab66340) mouse monoclonal Anti-Glucose-6-phosphate isomerase antibody. Glycogen phosphorylase was detected using the Abcam (ab88078). Monoclonal Anti-α-Tubulin antibody (Sigma–Aldrich) probed with the Polyclonal Rabbit Anti-mouse Immunoglobulins/HRP secondary antibody (Dako Cytomation) were used for the determination of the loading controls.
Ab88078
Manufactured by Abcam
Ab88078 is a laboratory product offered by Abcam. It is a tool for use in scientific research. The core function of this product is to provide a specific capability or component for experimental procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab76959, by Abcam (1 mentions)
Ab66340, by Abcam (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Monoclonal anti α tubulin antibody, by Merck Group (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Ab92696, by Abcam (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ab40810, by Abcam (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
3 protocols using ab88078
1
Quantification of Glycolytic Enzymes
2
Protein Expression Quantification in Muscle Biopsies
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Protein samples from muscle biopsies were fractionated on SDS–9% PAGE and blotted with anti-β-F1-ATPase (1:1000), anti-Hsp60 (1:1000), anti-GAPDH (1:1000) and anti-PKM2 (1:1000) from [11 (link)], anti-IF1 (1:500) from [12 (link)], anti-LDH-A (1:1000), anti-GPD1 (1:1000) from [13 (link)], anti-PYGM (Abcam, ab88078; 1:1000), anti-PKM1 (Abcam, ab6191-5; 1:1000), anti phospho-PKM2 (Tyr105) (Cell Signaling, #3827; 1:1000), anti-PDHE1α (Invitrogene, 459400; 1:500), anti-phospho-PDHE1α (Ser293) (Abcam, ab92696; 1:1000), anti-PDK1 (Abcam, ab207450; 1:1000) and anti-β-actin (Sigma-Aldrich, A1978; 1:1000). Peroxidase-conjugated anti-mouse IgGs (Nordic Immunology; 1:3000) were used as secondary antibodies. The blots were revealed using the ECL® reagent (Amersham Pharmacia Biotech). The intensity of the bands was quantified using a Kodak DC120 digital camera and the Kodak 1D Analysis Software.
3
Protein Expression Analysis of Hippocampal Tissue
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The powdered hippocampal tissues were homogenized in ice-cold lysis buffer. Aliquots of the clarified homogenized liquid containing 75 μg of protein were denatured and analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad). The primary antibodies included rabbit monoclonal anti-Gys 1 (1:2000, Abcam, ab40810), mouse monoclonal anti-Gyp (1:2000, Abcam, ab88078), and mouse monoclonal anti-β-actin (1:2000, Sigma, A1978). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:4000, GeneScript) or goat anti-rabbit IgG (1:4000, GeneScript). Immunoblotting was detected by enhanced chemiluminescence (Amersham) and analyzed using an FR-200A Electrophoresis Image Analysis System (Furi, Shanghai, China). The values of Gys 1 and Gyp levels were normalized against the amount of β-actin obtained from the same sample.
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