MONMOUTH JUNCTION, N.J. — Authors Hikosaka et al describe research on nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) from red blood cells and its regulation of nicotinamide adenine dinucleotide. Detection of hemolytic anemia and splenomegaly in addition to reduced adenosine triphosphate (ATP) levels was noted in cells not containing Nmnat3. Glycolysis progression was stalled at the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step as a deficiency of Nmnat3 led to lack of co-enzyme NAD. LC-MS/MS metabolomics and stable isotope tracer analysis revealed the impact of reduced NAD quantity in RBCs on glycolysis and pentose phosphate pathway. For selective extraction & isolation of hemoglobin from erythrocytes, HemogloBind™ from Biotech Support Group is used.

Sample preparation of RBC samples involved separation of RBCs by Percoll gradient method. Monoclonal antibody anti-mice Nmnat3 against his tagged mouse Nmnat3 recombinant protein & spleen cells for hybridoma cells via the polyethylene glycol method was performed followed by western blotting of hybridoma cells, centrifugation and lysis. Western blot using a monoclonal antibody against Nmnat3 recombinant protein was from heart, skeletal and liver tissues. Staining of cells, scanning electron microscope analysis, cell sorting experiments are implemented followed by in vivo RBC assay. Samples are labeled with 13C glucose and metabolites are extracted. ATP, RBC, WBC, reticulocyte cell count was performed.

The cytoplasmic fraction consisted of hemolyzed samples obtained from the supernatant and ghost for membrane fraction. An alpha cellulose & microcrystalline cellulose column for depleting platelets and leukocytes from heparinized whole blood for an assay of RBC metabolic enzymes is described. Extraction of metabolites from RBCs and NAD related metabolites from whole blood by perchloric acid method for LC-MS/MS measurement is done. Measurement of metabolite levels involved using MS and HPLC, quantitative analysis software and separations using a column.

The article states “…Each sample was normalized by hemoglobin concentration at 20 µg/µl, and then hemoglobin was depleted using HemogloBind™”.

Characteristics of HemogloBind™
Has a high degree of specificity for hemoglobin binding up to 10 mg/ml
Applications in blood substitutes, enzyme recovery and analytical interferences
Removes hemoglobin from any species including human, sheep, bovine, goat, etc
Removes hemoglobin from organs, tissues.
Hemoglobin removal from red blood cell lysate for proteomics and biomarker drug discovery
The flow through fractions retain their enzymatic and biological activity
The flow through fractions is compatible with LC-MS, activity based protein profiling and proteomic studies.

For more information visit: HemogloBind™, Hemoglobin Depletion From Hemolyzed Serum/Plasma
http://www.biotechsupportgroup.com/node/75

About Biotech Support Group LLC

Biotech Support Group LLC is a leading provider of genomics and proteomics sample preparation products and enrichment reagent kits as well as integrated biotechnology services for life sciences research, biomarker and drug discovery. Based in New Jersey, it’s principal products include: AlbuSorb™ & AlbuVoid™ for albumin depletion,Cleanascite™ for lipid adsorption and clarification, HemogloBind™ & HemoVoid™ for hemoglobin removal, NuGel™ for functional & chemical proteomics, andProCipitate™ & ProPrep™ for nucleic acid isolation. For more information, go to http://www.biotechsupportgroup.com

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References

Hikosaka, Keisuke, et al. “Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocyte“Journal of Biological Chemistry(2014): jbc-M114.