Aldolase a

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Aldolase A is an enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. It is a key enzyme in the glycolytic pathway.

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4 protocols using aldolase a

Western Blot Analysis of Protein Levels

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Tissue homogenates/lysates were normalized (90 µg of total protein per lane) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (NuPAGE; 4–12% Bis Tris-gradient gels, Invitrogen, Carlsbad, CA), transferred to nitrocellulose membranes, and analyzed by immunoblotting [2 (link)] (four rats/group). A total of four gels (labeled Gel 1 –4) were used to resolve all of the samples collected. After transfer, membranes were cut into strips representing desired molecular weight ranges based on a protein standard loaded in the far left lane of each gel [denoted as molecular weight marker (MWM) in kDa], and probed for protein levels by Western blotting. Primary antibodies against cas-pase-1, GAPDH, aldolase-A, and hexokinase II (Santa Cruz Biotechnology, Dallas. TX) were used. Protein transfer was assessed by staining membranes with BLOT Fast Stain (G Biosciences, St. Louis, MO), and total protein levels determined by fluorescence (Odyssey, LI-COR, Lincoln, NE). Total signal from each lane was used to normalize all immunoblot data.

Audia J.P., Yang X., Crockett E.S., Housley N., Ul Haq E., O’Donnell K., Cohen M.V., Downey J.M, & Alvarez D.F. (2018). Caspase‑1 inhibition by VX‑765 administered at reperfusion in P2Y12 receptor antagonist‑treated rats provides long‑term reduction in myocardial infarct size and preservation of ventricular function. Basic research in cardiology, 113(5), 32.

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Antibody Procurement for Research

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Antibodies in the present study were purchased from the following companies: V5 and green fluorescent protein (GFP) (Santa Cruz Biotechnology); aldolase A, GS, hexokinase II (HKII), Hsp70 and Hsp90, LC3A/B, pyruvate kinase M2 isoform (PKM2), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)2 and PFKFB3 (Cell Signaling Technology); p62, PYGL, pyruvate dehydrogenase kinase 1 (PDHK1), HMOX-1 and P62 (Proteintech). Bafilomycin A1 (BafA1) and chloroquine (CQ) were purchased from EMD Biosciences.

Yang R., Zhang M., Gustafson A.R., Wang E., Cole M.P., Tooley C.E, & Cheng A. (2015). Loss of protein targeting to glycogen sensitizes human hepatocellular carcinoma cells towards glucose deprivation mediated oxidative stress and cell death. Bioscience Reports, 35(3), e00207.

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Western Blot Analysis of Cell Proteins

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Whole-cell extracts (5~30 μg) were separated by 8–12% SDS-polyacrylamide gel electrophoresis, under reducing conditions and transferred onto nitrocellulose membranes (Amersham, Inc., Arlington Heights, IL, USA) by electroblotting. Protein transfer was verified using reversible staining with Ponceau S. Membranes were blocked with 3% non-fat dry milk in TBS-T (Tris-buffered saline and 0.2% Tween 20) for 1 h at room temperature. Proteins were detected using antibodies against vATPase E subunit (#PA5-29899; Thermo Fisher Scientific, Rockford, IL, USA), GPR120 (H-155; sc-99105; Santa Cruz Biotechnology, Dallas, TX, USA), GPR40 (SAB4501280, Sigma-Aldrich), aldolase A (sc-12059, Santa Cruz Biotechnology), Na⁺/K⁺-ATPase (sc-21712, Santa Cruz Biotechnology), and actin (sc-1615, Santa Cruz Biotechnology). Membranes were incubated overnight at 4 °C with primary antibodies diluted in TBS-T containing 3% non-fat dry milk. After washing with TBS-T, primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, anti-mouse, or anti-goat) and visualized using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology) after exposure to BioMax MR film (Kodak, Rochester, NY, USA). The target protein levels were compared to the levels of the loading control, actin.

Park Y., Ku L., Lim J.W, & Kim H. (2020). Docosahexaenoic acid inhibits zymogen activation by suppressing vacuolar ATPase activation in cerulein-stimulated pancreatic acinar cells. Genes & Nutrition, 15, 6.

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Western Blot Analysis of Cellular Extracts

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Whole cell extracts, membrane extracts, cytosolic extracts, and nuclear extracts were prepared as described previously [28 ]. 100–200 μg of protein was loaded per lane, separated by 8–12% SDS-polyacrylamide gel electrophoresis under reducing conditions, and transferred onto nitrocellulose membranes (Amersham, Inc., Arlington Heights, IL, USA) by electroblotting. The transfer of protein was verified using reversible staining with Ponceau S. Membranes which were blocked using 3% nonfat dry milk. The proteins were detected with antibodies for β-catenin, c-myc, p47, p67, NOX-1, aldolase A, histone H1, and actin (all from Santa Cruz Biotechnology) dilution in TBS-T containing 3% dry milk, and incubated overnight at 4°C, followed by secondary antibodies (anti-goat, anti-mouse, or anti-rabbit) conjugated to horseradish peroxidase and determination of enhanced chemiluminescence (Amersham) using exposure to BioMax MR film (Kodak, Rochester, NY) [30 (link)].

Byun E., Lim J.W., Kim J.M, & Kim H. (2014). α-Lipoic Acid Inhibits Helicobacter pylori-Induced Oncogene Expression and Hyperproliferation by Suppressing the Activation of NADPH Oxidase in Gastric Epithelial Cells. Mediators of Inflammation, 2014, 380830.

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