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HemoTrial™ Kit - 5 mL Hemoglobind™, 5 Preps of NuGel HemogloBind™, 5 Preps HemoVoid™
HemoTrial™ Kit - 5 mL Hemoglobindâ„¢, 5 Preps of NuGel HemogloBindâ„¢, 5 Preps HemoVoidâ„¢
 
 
Base Price: $580.00


Product Code: HTK


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HemoTrial™ Kit includes 5 ml of HemogloBind™ and 5 Preps of HemoVoid™. HemoVoid™, a silica-based protein enrichment matrix, removes hemoglobin from erythrocyte lysate samples while concentrating low abundance, and/or low molecular weight proteins. The HemoVoid™ protocol uses mild buffers; the protocol conditions are so gentle that native enzyme activity is retained in elution fractions. Poly-electrolytes are polymers with repeating units of stationary charges. HemogloBind™ comes from a class of solid-phase, or surface-based, elastomeric poly-electrolytic surfaces that bind proteins through an empirically derived chemistry combining elements of polymer composition, cross-linking architecture and charge properties. As with bio-polymers like DNA and Heparin, governing their reactivity is the spatial presentation of the electrostatic groups along a flexible polymer chain.

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References

Featured HemogloBind ™ Reference Applications –

Nguyen, Anthony T., et al. "UBE2O remodels the proteome during terminal erythroid differentiation." Science 357.6350 (2017): eaan0218.

During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of hemoglobin. The study used multiplexed quantitative proteomics to identify candidate substrates of UBE2O, an E2 (ubiquitin-conjugating) enzyme, in an unbiased and global manner. Because of the overly abundant presence of Hemoglobin, selective depletion of Hemoglobin was necessary. The article states “Reticulocytes were lysed by vortexing for 5 minutes at room temperature… An additional 10 bed vol of Hemoglobind™ suspension was added to the samples, which were then vortexed for another 10 min at room temperature followed by 4 min of centrifugation at 10000 x g. The supernatants, which contain hemoglobin-depleted sample, were … processed for TMT quantification.”.

Hemolyzed Serum Exosome Analyses

Nishida-Aoki, Nao, et al. "Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis." Molecular Therapy 25.1 (2017): 181-191.

http://dx.doi.org/10.1016/j.ymthe.2016.10.009

This study considers that therapeutic strategies targeting cancer-derived extracellular vesicles (EVs) hold great promise because of the possibility they reposition microenvironments to accommodate metastasis. The researchers report on a novel strategy of therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model. The article states “Hemoglobin was accumulated with HemogloBind™ beads (Biotech support group, Monmouth Junction NJ, USA) followed by 0.22 μm filtration. Then, the EVs in the sera were concentrated by ultracentrifugation…”.

Macromolecular Complexes and Functional Integrity

C Wan, B Borgeson, S Phanse, F Tu, K Drew, G Clark, et al. Panorama of ancient metazoan macromolecular complexes. Nature Volume:525, Pages:339–344 Date published:(17 September 2015). doi:10.1038/nature14877

HemogloBind™, contributed to this rigorous examination of protein complexes. When our products were used as a pretreatment step in the overall workflow, about twice the number of observations and annotations became possible. Furthermore, this study demonstrated the importance of a key feature implicit to all of our products; that is the maintenance of functional and structural integrity after separations. Without that particular feature, these additional observations would not have been possible.

Whole Blood

Lahut, Suna, et al. "Blood RNA biomarkers in prodromal PARK4 and REM sleep behavior disorder show role of complexin-1 loss for risk of Parkinson's disease." Disease Models & Mechanisms (2017): dmm-028035. http://dmm.biologists.org/lookup/doi/10.1242/dmm.028035

In this study, Parkinson’s disease progression is investigated through the accumulation and aggregation of the lipid-binding SNARE complex component alpha-synuclein (SNCA) which underlies vulnerability and defines its stages. The authors studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation), to identify effects of SNCA gain-of-function as potential disease biomarkers. The article states “For protein extraction from the EDTA tubes, 300 μl blood were lysed with equal amount of 1% SDS-RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and one tablet Complete Protease Inhibitor Cocktail (Roche)] and sonicated for 10 sec. The blood lysates were rotated at 4 °C for 30 min and centrifuged at 4 °C for 30 min. The supernatants were depleted in hemoglobin content using a commercial kit (HemogloBind™) following the manufacturer’s instructions”.

Chalásová, Katarína, et al. "Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction." Experimental and Clinical Endocrinology & Diabetes (2017). https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0043-115009

Diabetic kidney disease, a common complication of both type 1 and type 2 diabetes, is associated with significant morbidity and mortality, and represents the most common cause of chronic kidney disease. The study hypothesized that protective pentose phosphate pathway action in diabetes might be compromised by limited intracellular availability of an active transketolase cofactor thiamine diphosphate (TDP). To evaluate the levels of thiamine tranporter proteins in whole blood, t he article states “ For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group) according to manufacturer’s instructions…”.

Species Agnostic – Applications to Different Species

Snider, Thomas H., Christina M. Wilhelm, Michael C. Babin, Gennady E. Platoff Jr, and David T. Yeung. "Assessing the therapeutic efficacy of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig."The Journal of Toxicological Sciences 40, no. 6 (2015): 759-775.

Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. A primary component of anti-organophosphorus therapy is an oxime reactivator to rescue inhibited acetylcholinesterases. For this, clinical signs of toxicity can be measured from blood cholinesterase [Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] activity utilizing a modified Ellman's method. Biotech Support Group’s unique solid-phase polymer for hemoglobin depletion, was used for pretreatment. The article states “Briefly, whole blood samples were treated with HemogloBind™ which interferes with the ChE activity assay due to spectral overlap.”


Craig, J. R., et al. "A comparison of the anatomical and gastrointestinal functional development between gilt and sow progeny around birth and weaning." Journal of animal science (2019).

Gilt progeny (GP) often have restricted growth performance and health status in comparison to sow progeny (SP) from birth. To better understand underlying mechanisms, the study aimed to compare differences in growth and development between GP and SP in the first 24 h after birth and in the peri-weaning period. Because serum samples were quite hemolysed after collection and processing, it became necessary to use HemogloBind™ to allow for better detection of IgG by ELISA. The article states “As per the manufacturer’s instructions, 250 μL of Hemoglobind was added to 250 μL of hemolyzed serum…

Parvathi S. Kumar, Haree K. Pallera, Pamela S. Hair, Magdielis Gregory Rivera, Tushar A. Shah, Alice L. Werner,Frank A. Lattanzio, Kenji M. Cunnion, and Neel K. Krishna. Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats.TRANSFUSION Volume 00, May 2016. doi:10.1111/trf.13674.

In brief, the study evaluated the role of a peptide inhibitor of complement C1 (PIC1) in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios. The authors state "To remove free Hb that may cause optical interference in bilirubin analysis, we treated all the samples with Hb depletion from hemolyzed serum/plasma (HemogloBind, Biotech Support Group). Bilirubin concentration was then measured with a Bilirubin Assay Kit (Sigma-Aldrich, St. Louis, MO)."

Other References

Blood Substitutes

Laing, Richard W., et al. "The use of an acellular oxygen carrier in a human liver model of normothermic machine perfusion." Transplantation 101.11 (2017): 2746.

Red cell lysates
O’Connell, Grant C., et al. "Monocyte-lymphocyte cross-communication via soluble CD163 directly links innate immune system activation and adaptive immune system suppression following ischemic stroke." Scientific reports 7.1 (2017): 12940

Kyoungsook Park, Christopher D. Saudek, and Gerald W. Hart Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals.Diabetes.2010;59(7):1845-50.

Delobel J., Rubin O., Prudent M., Crettaz D., Tissot J.-D., Lion N.(2010) Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues. International Journal of Molecular Sciences. 11(11):4601-4617

Alvarez-Llamas, Gloria, Fernando de la Cuesta, Maria G. Barderas, Irene Zubiri, Maria Posada-Ayala, and Fernando Vivanco. "Characterization of Membrane and Cytosolic Proteins of Erythrocytes." In Vascular Proteomics, pp. 71-80. Humana Press, 2013.

Alvarez-Llamas, G., de la Cuesta, F., Barderas, M. G., Darde, V. M., Zubiri, I., Caramelo, C., Vivanco, F. A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE.Electrophoresis.2009;30:4095-4108

Zihao Wang, Kyoungsook Park, Frank Comer1, Linda C. Hsieh-Wilson, Christopher D. Saudek, Gerald W. Hart. Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus. Diabetes.2008;58, 309-317.

Yuichi Miki, Tomoki Tazawa, Kazuya Hirano, Hideki Matsushima, Shoko Kumamoto, Naotaka Hamasaki, Tomohiro Yamaguchi, Masatoshi Beppu. Clearance of oxidized erythrocytes by macrophages: Involvement of caspases in the generation of clearance signal at band 3 glycoprotein. Biochemical and Biophysical Research Communications.2007; 363(1):57-62

Sarawathi,et al., Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry, Biochimica et Biophysica Acta. 1999.1426(3):483-90


Featured HemoVoid™ Reference Applications

Human Red Blood Cells (RBC)

Bollenbach, Alexander, et al. "GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells." Journal of Chromatography B (2020): 122024.

Previous studies showed that human red blood cells are rich in large (> 50 kDa) asymmetric dimethylarginine -containing proteins of unknown identity. The study aimed to report the identity, biological activity and concentration of NG-methylated proteins by using GC-MS and LCMS/MS approaches. The article states “we included in our method the use of HemoVoid™ to remove specifically most erythrocytic hemoglobin and to improve the SDS-PAGE separation of proteins for further processing. The HemoVoid™, … allowed removal of erythrocytic hemoglobin to a large extent from the hemolysate. … removal of hemoglobin by this technique enabled an effective separation by SDS-PAGE and isolation of bands, presumably by avoiding overloading of the gels by hemoglobin.”.

Kitao, Akihito, et al. "Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia." International Journal of Hematology (2020): 1-10.

Autoimmune hemolytic anemia (AIHA) is a rare comorbidity in colorectal cancer (CRC) and has an unknown etiology. To better understand cancer-related anemia, the authors’ investigated ectopic band 3 expression and erythrocyte membrane-bound IgG in a CRC cohort. To reduce the interference from Hemoglobin, the article states “Erythrocytes were lysed … and hemoglobin was depleted using HemoVoid (Biotech Support Group, NJ, USA, Cat. No. HVK-10)”.

Rosin-Arbesfeld, Rina, and Ronen SIMAN-TOV. "Article of manufacture and methods for increasing survival of red blood cells." U.S. Patent Application No. 15/739,857.

The patent application describes an ex - vivo method of increasing survival of red blood cells (RBCs). The method comprises contacting the RBCs with an activator of the non - canonical Wnt pathway, which results in actin polymerization, thereby increasing survival of RBCs. The invention’s description states “The Haemolysates were enriched with over 95 % hemoglobin. For hemoglobin depletion, the hemoglobin depletion kit of HemoVoid … was used”. Upon depletion of hemoglobin, a reduction in cytoplasmic actin levels was observed.

Nemkov, Travis, et al. "Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage." haematologica 103.2 (2018): 361-372.

The goal of this study was to use proteomics in part to understand hypoxanthine catabolism in vivo for stored red blood cells. It is still unclear whether accumulation of hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. The article states “Leukocyte-reduced human RBC from healthy donor volunteers were washed five times in phosphate-buffered saline prior to lysis in distilled water with sonication. Proteomic analyses of RBC membranes and cytosols were performed…RBC cytosolic proteins were depleted of hemoglobin using Hemovoid™ (Biotech Support Group, Monmouth Junction, NJ, USA), prior to high-pH reversed phase fractionation”.

Cortese-Krott, Miriam M., et al. "Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease." Redox Biology (2017).

http://www.sciencedirect.com/science/article/pii/S2213231717306535


In brief, the authors aimed to investigate whether RBCs carry a functional soluble guanylate cyclase (sGC) signalling pathway and to address whether this pathway is compromised in coronary artery disease. The article states “Using a commercial resin (HemoVoid™), which removes hemoglobin… and allows enrichment of soluble cytoplasmic proteins, we established a procedure that allows fast and reliable preparation of hemoglobin-free cell lysates from as little as 1-2 ml blood. In those samples, expression and activity of the cGMP-generating sGC, cGMP-hydrolyzing PDE5 and cGMP-transducing PKG was assessed by enzymatic assays and Western blot analysis”.

Feliciano, Amélia, et al. "Evening and morning alterations in Obstructive Sleep Apnea red blood cell proteome." Data in Brief (2017). http://dx.doi.org/10.1016/j.dib.2017.01.005

Using proteomics-based evaluation of red blood cells (RBC), the authors identified differentially abundant proteins associated with Obstructive Sleep Apnea Syndrome (OSA). Proteome variations between various time points were assessed. The article states “RBC cytoplasmic fraction depleted of hemoglobin, using HemoVoid™ system, were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), the 2D image software-based analyzed and relevant differentially abundant proteins identified by mass spectrometry (MS)”.

Philipp F Lange, Pitter F Huesgen, Karen Nguyen, and Christopher M Overall. ” Annotating N termini for the Human Proteome Project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome”, J. Proteome Research., Just Accepted Manuscript • DOI: 10.1021/pr401191w • 21 Feb 2014

The article describes a goal of the Chromosome-centric Human Proteome Project to identify all human protein species. Enucleated, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content (about 97% by mass) and wide dynamic range of protein concentrations that impedes protein identification. Using a N-terminomics procedure called TAILS, the authors identified from the HemoVoid™ treated, soluble fraction, 778 proteins were identified, 171 of which were not represented in either the soluble non-depleted fraction or the membrane fraction.

Barasa, Benjamin, and Monique Slijper. "Challenges for red blood cell biomarker discovery through proteomics." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844.5 (2014): 1003-1010.

Katja Walpurgis, Maxie Kohler, Andreas Thomas et al. Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D-PAGE and Orbitrap MS.Electrophoresis.2012;

Mizukawa, B., George, A., Pushkaran, S. et al. Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis.Pediatric Blood Cancer.2011;56: 840-842.

Sudha Neelam, David G Kakhniashvili, Stephan Wilkens et al. Functional 20S proteasomes in mature human red blood cells Experimental Biology and Medicine.2011;236:580-591

HemoVoid™ On Bead Digestion Application Work On RBC by Irene Granlund, Umeå University

RBCs in Parkinson’s Disease

Klatt, Stephan, et al. "Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry." Analytical and Bioanalytical Chemistry (2020): 1-14.

The article describes the advantage of HemoVoid™ in detection of low abundance proteins when comparing their amounts (in percent) between four alternative extraction conditions, stating “… Most peptides, following HemoVoid™ extraction, showed ion abundances ranging between 1.00E+5 and 1.00E+6 (31%). In comparison to this, fewer peptides (10–23%) were within this range following extraction with all other protocols”. With respect to potential biomarkers for Parkinson’s Disease, the article states “For example, PRDX6 accounts for 0.4% of the total ion abundance after DOC (deoxycholate) extraction, whereas following HV (HemoVoid™) extraction, this increases to 8%, a 20-fold enrichment”. The authors conclude that the HemoVoid™ method significantly reduces the concentration of hemoglobin, resulting in an increased signal-to noise of the remaining red cell proteins. The article describes methods to digest the HemoVoid™ bead-bound proteome, greatly simplifying the workflow for LC-MS/MS analysis.


Elhadi, Suaad Abd, et al. "α‐Synuclein in blood cells differentiates Parkinson’s disease from healthy controls." Annals of Clinical and Translational Neurology.

The goal of this study was to determine whether blood cells expressing α-Synuclein can differentiate Parkinson’s disease (PD) from healthy controls. Two proteoforms - PSer129 a-Syn (phosphorylated pathological form in Lewy bodies) and Oxidized a-Syn levels are observed in blood cells, but both at considerably lower concentration than total a-Syn, so the extremely high abundance of hemoglobin interferes with their analysis. To compensate, the article states for PSer129 α -Syn & Oxidized α -Syn detection by immunoassay, “followed from hemoglobin clearance with HemoVoid kit (Biotech Support Group LLC, NJ, US)”.

Red Blood Cells, Plasmodium extracts
Machado, Patrícia Isabel Pires. Pyruvate kinase and glucose-6-phosphate dehydrogenase deficiencies and their association with malaria–population genetics and proteomic studies. Diss. Universidade do Porto, 2013.

Walpurgis, Katja, et al. "Effects of gamma irradiation and 15 days of subsequent ex vivo storage on the cytosolic red blood cell proteome analyzed by 2D DIGE and Orbitrap MS." PROTEOMICS-Clinical Applications (2013).

P. Falciparum Clone 3D7 Cultured In Human Erythrocytes
Lasonder E, Green JL, Camarda G, Talabani H, Holder AA, Langsley G, Alano P. The Plasmodium falciparum schizont phospho-proteome reveals extensive phosphatidylinositol and cAMP-Protein Kinase A signalling. J Proteome Research. 2012;

Species Agnostic – Applications in non-human samples

Puente-Marin, Sara, et al. "In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling." Genes 9.4 (2018): 202.

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. Label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. The article states “The cytosolic fraction, approximately 300 μL, was depleted of hemoglobin using HemoVoidTM kit (Biotech Support Group, Monmouth Junction, NJ, USA), in accordance with the manufacturer’s instructions”.

Nombela I, Puente-Marin S, Chico V et al. Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication [version 1; referees: awaiting peer review].
F1000Research 2017, 6:1958 (doi: 10.12688/f1000research.12985.1)

Fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses replicate inside them and are their main target cell. However, the objective of this study not yet explored, was to determine the immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues. The article states “a new proteomic analysis method was carried out that combines fractionation into cytosolic and membrane fractions, haemoglobin removal of the cytosolic fraction, protein digestion, pH reversed-phase peptide fractionation and finally LC ESI-MS/MS analysis of each of the fractions… . Briefly, the haemoglobin of the cytosolic fraction was removed using a column of HemoVoid™ kit (Biotech Support Group, Monmouth Junction, NJ), following the manufacturer instructions”.