NuGel™ Related References
Affinity
Dermot Walls, Robert McGrath and Sinéad T.Loughran A Digest of Protein Purification. Methods Molecular Biology.2011;(681): 3-23
Authors cite NuGel™ as an ideal support for affinity purification. NuGel™'s application includes immobilization of proteins and ligands. This chapter reviews advances in protein chromatography and protein purification processes. For example, NuGel™-Epoxy has epoxy-activated supports which chemically react with nucleophile groups on protein surfaces such as lysine, histidine, cysteine, tyrosine etc. This chapter mentions NuGel™'s application for protein immobilization on supports before implementing chromatographic procedures on proteins for binding affinity, size and physico-chemical properties.
Ehrlich, G. K., Michel, H., Chokshi, H. P. and Malick, A. W. Affinity purification and characterization of an anti-PEG IgM. Journal of Molecular Recognition.2009;22: 99-103.
Scientists used PEG-derivatized NuGel™ to bind anti-PEG IgM for purifying anti-PEG IgM. The ligands consisted of variable length PEG chains (5, 10, 20 and 30 kDa). After purifying anti-PEG IgM via affinity chromatography the PEG-derivatized NuGel™ underwent pepsin digestion. Subsequent analysis of anti-PEG IgM and fragments occurred using PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Such studies create probes for assays used in funcationalized PEG molecules.
Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: New particulate Lyme disease vaccines. International Journal of Medical Microbiology. 2008; 298(S1-2):135-142
NuGel™ Poly-Amine(Matrix Reactive Group: Terminal Amine) is used for the immunogenicity of peptides and small protein fragments can be considerably enhanced by the presentation on particulate carriers such as capsid-like particles (CLPs) from hepatitis B virus (HBV). NuGel™'s unique reversed phase supports have been developed for large scale purification of biological macromolecules (i.e. proteins, peptides, DNA, RNA, etc)
A sensitive and high-throughput assay to detect low-abundance proteins in serum Hongtao Zhang, Xin Cheng, Mark Richter & Mark I Greene. Nature Medicine 12, 473 - 477 (2006)
Scientists used NuGel™ to increase stability of antibodies and antigens binding immunologically while developing fluorescent amplification catalyzed by T7 polymerase technique (FACTT) for amplification of detecting protein targets at subfemtomolar levels (~0.08 fM). Ultimately such techniques are high throughput compatible and used for screening and automationof ELISA.
Transformation of a L-peptide epitope into a D-peptide analog. Peptides Frontiers of Peptide Science American Peptide Symposia.2002;5:769-770
In vivo proteases often cause degradation of short synthetic peptodes used as vaccines or therapeutic agents. Scientists used NuGel™ for it's specificity and binding affinity to increase stability while transforming L-amino peptides into D-amino acid analogs.
Ehrlich, G. K., and P. Bailon. "Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display." Journal of Biochemical and Biophysical Methods 49.1-3 (2001): 443-454.
Phage-display technology for tagging peptide leads in the customization of ligands for NuGel™ Poly-Amine(Matrix Reactive Group: Terminal Amine) based affinity chromatography allowed scientists to identify a peptides in the affinity purification of monoclonal antibodies. The Amino-NuGel™ (500 A, 50-60um) from Biotech Support Group is used to immobilize the synthetic peptides.
George K. Ehrlich, Pascal Bailon, Wolfgang Berthold. Phage Display Technology - Identification of Peptides as Model Ligands for Affinity Chromatography Affinity Chromatography Methods in Molecular Biology.2000;147:209-220
In the procedure for screening of phage peptide libraries against monoclonal antibodies, scientists used NuGel™ for coupling peptide mimotope to the affinity matrix for purifying antibodies from human serum. Producing peptides on bacteriophages requires selection for ligand-binding molecules ie selection against target molecules and tailoring ligands to numerous applications. Authors Ehrlich et al describe the selection of peptide ligands for affinity chromatography (especially peptides that mimic antigen epitopes).
A Digest of Protein Purification and partial amino acid sequence of a 28 kDa cyclophilin-like component of the rat liver sigma receptor. Life Sciences. Volume 55, Issue 8, 1994.
Researchers used NuGel™ to eliminate non specific sites and for coupling ligands during receptor purification of amino acid sequence identical to the N-terminal sequence of the 17 kDa rat cyclophilin A, a cytosolic protein. Nugel was used during the purification of the non-opiod sigma receptor which functions as a neuromodulator of dopaminergic and NMDA systems in brain, liver and organ tissues. The protein was purified NuGel™, affinity matrix from ocimino derivatives of haloperidol by affinity chromatography.
Nachman, M., Azad, A. R. M. and Bailon, P. Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC). Biotechnology and Bioengineering.1992;40: 564-571.
Design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins. NuGel™ Poly-Amine(Matrix Reactive Group: Terminal Amine) is used in the preparation and characterization of immunoaffinity membranes using immunoaffinity purification process for recombinant interferon-alpha2a. Using NuGel's membrane-based immunoaffinity chromatography human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, are purified efficiently.
Kinetic aspects of membrane-based immunoaffinity chromatography. Journal of Chromatography A.1992; 597:(S1-2):167-172
NuGEL™ Poly-Amine(Matrix Reactive Group: Terminal Amine) is used in a study for membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, for the purification of IL2. The affinity membrane had a interleukin-2 receptor (IL-2R) in a model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) to determine factors affecting MRAC, including support morphology, mass transfer rate and adsorption kinetics.
Membrane-based receptor affinity chromatography. Journal of Chromatography A.1992;597(S1-2):155-166
9th International Symposium on Affinity Chromatography and Biological Recognition
In Purification and Analysis of Recombinant Proteins authors Seetharam and Sharma cite NuGel™ affinity support immobilization which allows for efficient receptor coupling and recovery of binding capacities. Activated polymer support forms stable covalent bonds with sequence of reagents when mixed with proteins. Authors recommend NuGel™ for industrial scale operations because of mechanical rigidity and flow rates which provide a durable bed support resulting in four fold flux than compared to agarose supports. Authors present the use of receptor affinity chromatography of recombinant interleikun 2 for the purification of recombinant proteins.
Schuster DI, Ehrlich GK, Murphy RB.Purification and partial amino acid sequence of a 28 kDa cyclophilin-like component of the rat liver sigma receptor.Life Sci. 1994;55(8):PL151-6.
Scientists used NuGel™ Poly-Amine(Matrix Reactive Group: Terminal Amine) to study the sigma (σ) receptor as a neuromodulator of dopaminergic and NMDA systems found in brain, liver. NuGel™ allowed for purification from a detergent-solubilized rat liver membrane preparation by using the affinity media chromatography and a affinity matrix prepared from an oximino derivative of haloperidol. The principle components are retained in the NuGel™ affinity column and are eluted from the column with dextrallorphan and haloperidol. After dialysis and concentration by ultrafiltration, scientists predicted that rat cyclophilin A, a cytosolic protein is a critical component of the rat liver σ receptor. These results support the suggestion that σ receptors are a key link between the central nervous system and the immune system.
Patents
Purification of immunoglobulins using affinity chromatography and peptide.
NuGel™ Poly-Amine(Matrix Reactive Group: Terminal Amine) is used for the purification of human immunoglobulins (Igs), a class of plasma proteins produced by the immune system. It is divided into five classes, IgG, IgA, IgM, IgD, and IgE. Monoclonal antibodies produced in cell culture from hybridoma cells is a source for the production of HIgG. This patent mentions the advantage of using affinity chromatography because it allows for several fold purification with high recovery in a single step. Protein A and protein G are the most commonly used ligands in the purification of HIgG.
Cytotoxic lymphocyte maturation factor 40kD subunit and monoclonal antibodies directed thereto
In this article, a supernatant solution is passed through a Ion-Exchange Chromatography on NuGel™ P-SP Column (5cm x 5cm) equilibrated in 10mM MES, pH 6.0. The column was washed until baseline absorbance monitoring at 280 nm was obtained. Next the absorbed proteins are eluted with a 500 ml salt gradient from 0 to 0.5 M NaCl/10 mM MES, pH 6.0 at a flow rate of 2 ml/min (Fig. 1). Aliquots of fractions were assayed for T cell growth factor (TGF) activity. Fractions containing TGF activity were pooled and dialyzed against 50 volumes 20 mM Tris/HCl, pH 7.5 in order to reduce the salt concentration of the preparation.
Ion Exchange
Levin W Protein Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture. Protein expression and purification.1992 3(1):27-35.
Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 μg/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (>65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1–4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8–14.2 kDa) over a protein concentration range of 10 μ/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.
Patents
Monoclonal antibodies directed to the cytotoxic lymphocyte maturation factor European Patent EP0790255
The invention displays antibodies which bind to a novel cytotoxic lymphocyte maturation factor. When bound to the cytotoxic lymphocyte maturation factor, the antibodies can neutralize bioactivity of the factor. For the Purification and Characterization of Cytotoxic Lymphocyte Maturation Factor (CLMF) scientists used ion-Exchange Chromatography on NuGel™ P-SP Column.
Purification of immunoglobulins using affinity chromatography and peptide USA 2006/0153834 A1
Authors cited NuGel™-amino as a ligand used in the purification of IgG. Isolated HIgG form multimers as it is obtained by the Cohn-Oncley process from pooled plasma or from monoclonal antibodies from hybridoma cells. Diafiltered supernatant of cell culture is purified using ammonium sulfate precipitation, ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), or affinity chromatography. The EPIHRSTLTALL ligand serves as an alternative to Protein and Protein G which have high cost and low stability. Affinity chromatography provides stable high recovery and NuGel™ provides stable support for wide pH range 2 – 10 with 1000Å, 50µm silica suitable for LC and batch processes.
