Aldoa

Manufactured by Cell Signaling Technology

Sourced in United States

ALDOA is a protein that catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. It is an essential enzyme in the glycolytic pathway.

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Anti-ALDOA

5 protocols using aldoa

Affinity Capture of Biotinylated Proteins

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For immunoprecipitation experiments, the total cell lysate from cells incubated with butenylated biotin, biotinylated MSPA-961, or biotinylated MSPA-735 and then incubated with specific antibody overnight at 4 °C with a constant rotation, followed by the pull-down of the immune complex with streptavidin–agarose beads (Invitrogen, Waltham, MA, USA). The immunoprecipitated proteins were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween (TBS-T) buffer for 60 min. Primary antibodies against ALDOA, GAPDH, ENO1, and FH (Cell Signaling Technology, Danvers, MA, USA) were used individually at 1:1000 dilutions in 5% non-fat milk in TBS-T buffer for 90 min, and then washed with TBS-T buffer. Membranes were incubated with anti-rabbit secondary antibody (1:4000) in 5% non-fat milk in TBS-T buffer for 30 min and then washed in TBS-T buffer for 30 min. The bands were visualized using horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA).

Song K., Rajasekaran N., Chelakkot C., Lee H.S., Paek S.M., Yang H., Jia L., Park H.G., Son W.S., Kim Y.J., Choi J.S., Jeong H.M., Suh Y.G., Yun H, & Shin Y.K. (2021). Macrosphelide A Exhibits a Specific Anti-Cancer Effect by Simultaneously Inactivating ENO1, ALDOA, and FH. Pharmaceuticals, 14(10), 1060.

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Western Blot Analysis of Cellular Proteins

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Cell lysis was performed at 4 °C using ice cold RIPA lysis buffer containing 25 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS. Protein extracts were analysed by SDS-PAGE and Western blotting. ECL signals were detected as described before [76 (link)]. The following antibodies were used: α-tubulin (Thermo Fischer Scientific, Waltham, MA, USA), CD63 (Merck Millipore, Burlington, MA, USA), ALDOA, calnexin, FXR2, PKM2, PRMT5 (Cell Signaling, Danvers, MA, USA), CD81 (Biolegend, San Diego, CA, USA), HIF1α (BD Biosciences, Franklin Lakes, NJ, USA), CD9, Tsg101 (Abcam, Cambridge, UK), IPO11 (Novus Biologicals, Centennial, CO, USA).

Walbrecq G., Lecha O., Gaigneaux A., Fougeras M.R., Philippidou D., Margue C., Tetsi Nomigni M., Bernardin F., Dittmar G., Behrmann I, & Kreis S. (2020). Hypoxia-Induced Adaptations of miRNomes and Proteomes in Melanoma Cells and Their Secreted Extracellular Vesicles. Cancers, 12(3), 692.

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Western Blot Analysis of Cellular Proteins

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Total protein was isolated from cells using Cell Extraction Buffer (Biosource, Camarillo, CA) supplemented with protease and phosphatase inhibitors and precleared using centrifugation, followed by measuring protein concentrations using the BCA Protein Assay kit (Pierce, Rockford, IL). The primary antibodies including TAZ, CTGF, Notch1, Hes1, LDHA, PFKB3, PEPCK, PKM2, PGK1, HK2, GLUT1, GLUT3, ALDOA, p-TAZ (Ser89), and β-actin were purchased from Cell Signaling Technology (CA, US). Jagged1 and TEAD1 were purchased from Santa Cruz Biotechnology (CA, US). Appropriate secondary antibodies conjugated to horseradish peroxidase were used, including anti-mouse or anti-rabbit IgG (GE-Healthcare, CA, US). Proteins were visualized by Amersham enhanced chemiluminescence (GE-Healthcare, CA, US). Immunohistochemistry assay was performed as previously described [7 (link)].

Xie M., Fu X.G, & Jiang K. (2021). Notch1/TAZ axis promotes aerobic glycolysis and immune escape in lung cancer. Cell Death & Disease, 12(9), 832.

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Glycolytic Enzyme Immunoblotting Assay

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Samples were denatured in SDS loading buffer and heated at 95°C for 5 minutes. Proteins were then resolved via SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Piscataway, NJ). Membranes were blocked with Odyssey Blotting Buffer (Li-Cor Biosciences, Lincoln, NE) for 45 minutes at room temperature. Primary antibodies were incubated with membranes overnight at 4°C as described: GLO1 (1:2000, MilliporeSigma, 05-1925), GLO2 (1:1000, ThermoFisher, PA5-28292), HK-1 (1:2000, Cell Signaling Technologies, #2024), LDHA (1:2000, Cell Signaling Technologies, #3582), PKM1/2 (1:2000, Cell Signaling Technologies, #3190), ALDOA (1:2000, Cell Signaling Technologies, #8060). Following 3x washes with TBS +0.1% Tween-20, infrared secondary antibodies (Li-COR) were added in blocking buffer (1:5000) for 45 minutes. Blots were developed following 3 additional washes with TBST using a c600 Azure Imaging System (Azure Biosystems, Dublin, CA).

Gaffney D.O., Jennings E.Q., Anderson C.C., Marentette J.O., Shi T., Oxvig A.M., Streeter M.D., Johannsen M., Spiegel D.A., Chapman E., Roede J.R, & Galligan J.J. (2019). Non-Enzymatic Lysine Lactoylation of Glycolytic Enzymes. Cell chemical biology, 27(2), 206-213.e6.

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Metabolic Reprogramming in C. tropicalis

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The C. tropicalis strain (W4162870) was kindly provided by Dr. Sarah L. Gaffen (University of Pittsburgh, PA). Oxaliplatin(S1224), Forskolin (S2449) and Sodium Oxamate (S6871) were purchased from Selleck Chemicals (Houston, Texas, USA). Lactate was purchased from Sigma (St. Louis, MO, USA). Antibody against MLH1 (#3515), MSH2 (#2017), Cleaved caspase3 (#9664), caspase9 (#9508), p-H2AX (Ser139, #9718), PFKFB3 (#13123), LDHA (#3582), PKM2 (#4053), PGAM1 (#12098), GPI (#57893), GLUT1(#12939), ALDOA (#8060), HK2(#2867), PKAC-α(#4782) and p-CREB (Ser133, #9198)were purchased from Cell Signaling Technology (Boston, MO, USA). Antibody against β-tublin (# 66240-1-Ig) was purchased from Proteintech (Wuhan, Hubei, P.R.C). Antibody against GPR81 (YN2554) was purchased from Immunoway (Plano, TX, USA). CCK8 Kit (CK04) was purchased from Dojindo Laboratories (Japan). Annexin V FITC/PI Apoptosis Kit (A211-01) was purchased from Vazyme Biotech (Nanjing, China). F4/80-APC (#123115), CD11b-FITC (#301330), Ly6G-PE (#127607), Ly6C-APC (#128016), Gr-1-FITC (#108405) were all from Biolegend (San Diego, CA, USA). Lactate Assay Kit I (#120001100P) and Glucose Assay Kit I (#120003100P) were purchased from Eton Bioscience (San Diego, USA). Cyclic AMP Complete ELISA Kit (ab133051) was purchased from abcam (Cambridge, England).

Qu J., Sun Z., Peng C., Li D., Yan W., Xu Z., Hou Y., Shen S., Chen P, & Wang T. (2021). C. tropicalis promotes chemotherapy resistance in colon cancer through increasing lactate production to regulate the mismatch repair system. International Journal of Biological Sciences, 17(11), 2756-2769.

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