Milliplex map human glycolysis pathway magnetic bead panel hgpmag 27k

The MILLIPLEX MAP Human Glycolysis Pathway Magnetic Bead Panel (HGPMAG-27K, Millipore, Billerica, MA) was applied in 96-well plates for the quantification of GPI/AMF in tissue lysates. For the immunoassay procedures, 25 μl of each dilute lysate sample in assay buffer (5 μg total protein/well) and HeLa cells lysate (positive control) were added into wells in duplicate, according to the manufacturer's instructions. To each well, 25 μl of the mixed beads was added and the plate was incubated for 2 hours at room temperature. Human glycolysis pathway detection biotinylated antibodies were added for 1 hour; each captured a specific bead. After that, the reaction mixture was incubated for 30 minutes with streptavidin–PE conjugate to complete the reaction on the surface of each microsphere. Finally, the MILLIPLEX MAP was analyzed by Luminex xMAP technology. The immunoassay on the surface of each fluorescent-coded magnetic bead, MagPlex-C microsphere, was identified and quantified based on fluorescent signals. The median fluorescence intensity (MFI) was read with the Luminex 200 instrument and measured with xPONENT software.

The MILLIPLEX^® MAP Human Glycolysis Pathway Magnetic Bead Panel (HGPMAG-27K, Millipore) was applied in 96-well plates for the simultaneous quantification of the following proteins in tissue lysates: G6PI/AMF (Glucose-6-phosphate isomerase/Autocrine motility factor), LDHA (L-lactate dehydrogenase A chain), LDHB (L-lactate dehydrogenase B chain), PKM2 (Pyruvate kinase isoform M2) and TKT (Transketolase), and HIF-1α (Hypoxia-inducible factor 1-α).
For the immunoassay procedures, 25μl of each dilute lysate sample in Assay Buffer (5μg total protein/well) and HeLa cells lysate (positive control) were added into wells in duplicate, according to the manufacturer's instructions. To each well, 25 μl of the Mixed Beads were added and the plate was incubated for 2 hours at room temperature. Human glycolysis pathway detection biotinylated antibodies were added for 1 hour; each captured a specific bead. After that, the reaction mixture was incubated for 30 minutes with Streptavidin-PE conjugate to complete the reaction on the surface of each microsphere. Finally, the MILLIPLEX^® MAP was analyzed by Luminex xMAP^® technology. The immunoassay on the surface of each fluorescent-coded magnetic bead, MagPlex-C microsphere, was identified and quantified based on fluorescent signals. The median fluorescence intensity (MFI) was read with the Luminex 200^TM instrument and measured with xPONENT^® software.

The MILLIPLEX^® MAP Human Glycolysis Pathway Magnetic Bead Panel (HGPMAG-27K, Millipore, Burlington, MA, USA) was applied in 96-well plates for the simultaneous quantification of the following proteins in tissue lysates: G6PI (glucose-6-phosphate isomerase), LDHA (L-lactate dehydrogenase A chain), LDHB (L-lactate dehydrogenase B chain), PKM2 (pyruvate kinase isoform M2), TKT (transketolase) and HIF-1α (hypoxia-inducible factor 1-α).
For the immunoassay procedures, 25 µL of each dilute lysate sample in assay buffer (5 µg total protein/well) and HeLa cell lysate (positive control) were added into wells in duplicate, according to the manufacturer’s instructions. To each well, 25 µL of the Mixed Beads were added and the plate was incubated for 2 h at room temperature. Human glycolysis pathway detection biotinylated antibodies were added for 1 h; each captured a specific bead. After that, the reaction mixture was incubated for 30 min with Streptavidin-PE conjugate to complete the reaction on the surface of each microsphere. Finally, the MILLIPLEX^® MAP was analyzed by Luminex xMAP^® technology. The immunoassay on the surface of each fluorescent-coded magnetic bead, MagPlex-C microsphere, was identified and quantified based on fluorescent signals. The median fluorescence intensity (MFI) was read with the Luminex 200TM instrument and measured with xPONENT^® software.