chromatography Protocols

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Antibody Purification (Antiserum or Ascites by Protein A/G Chromatography). Species and Type of Antibody Agarose Rabbit IgG Protein A or Protein G Mouse IgG1 Protein G Mouse IgG2 Protein A or Protein G Mouse IgG3 Protein G Sheep IgG Protein G but binds weakly Rat IgG Protein G but binds weakly Guinea Pig IgG Protein A Dog IgG Protein A Goat IgG Protein G Pig IgG Protein A Hamster IgG Protein G. By Millipore. - [Read Affinity Antibody Purification of Protein A/G Chromatography]

Category: Chromatography Protocols

Affinity Chromatography Handbook. Google Books. - [Read Affinity Chromatography Handbook]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol provides a quick way to assay for RNA-dependent RNA polymerase (RdRP) activity in crude protein samples.  RdRP transcription products remain at the origin during paper chromatography. - [Read An Assay for RNA-Dependent RNA Polymerase Activity Protocol]

Category: Molecular Biology Protocols: Carbohydrate Protocols

High performance liquid affinity chromatography (HPLAC) is a useful procedure to investigate he interactions between carbohydrate binding protein and their ligands. Technical requirements are similar to conventional HPLC. HPLAC can screen and separate natural ligands from complex biological mixtures. WeiTong Wang~GlycoTech Corporation, Rockville, Maryland - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

Category: HPLC Protocols

Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography Protocol. Glycotech. - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

Category: Protein Protocols: Antibody Purification Protocols

This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography. Mike A. Dyer Lab Harvard. - [Read Antibody Purification]

Category: Protein Protocols: Antibody Purification Protocols

Antibody Purification (Antiserum or Ascites by Protein A/G Chromatography). Buffer Preparation, Preparation of a Protein A Agarose or Protein G Agarose Affinity Column, Pouring the Protein A/G Affinity Column, Preparation of Antiserum or Ascites for Affinity Chromatography, Affinity Chromatography Using Protein A/G Agarose. - [Read Antibody Purification - Antiserum or Ascites by Protein A/G Chromatography]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

ANTIBODY PURIFICATION by affinity chromatography. By Beth, Mullins Lab UCSF. To affinity purify antibodies, generate lots of E. coli lysate that contains your antigen. If the protein can stand freeze thawing, then go ahead and purify the protein from e. coli lysate and keep it frozen until you need to couple it to a CH-sepharose column. - [Read ANTIBODY PURIFICATION by affinity chromatography]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions.  The amino acid histidine present in many proteins forms complexes with transition metal ions such as Cu2+, Zn2+, Ni2+ and Fe3+.  Chelating Sepharose™ Fast Flow with a suitable immobilized metal ion will therefore selectively retain proteins with exposed histidine. - [Read Chelating Sepharose Fast Flow Protocol]

Category: Chromatography Protocols: Column Chromatography Protocols

Size Exclusion Column Chromatography Protocol. PDF. In this protocol you will learn how to use three types of column chromatography: Gel Filtration or Size Exclusion (SEC), Ion Exchange (IEC), and affinity (AC) in order to purify proteins and enzymes based on the physical properties of these biomolecules. Univ. Arizona, Biochemistry. - [Read Column Chromatography Protocol]

Category: Chromatography Protocols: Column Chromatography Protocols

Column Chromatography Protocol DOC, includes troubleshooting information. Equipment used included: Peristaltic pump, UV detector , Chart recorder, and Column. Prestegard Lab. - [Read Column Chromatography Protocol DOC]

Category: Chromatography Protocols: Column Chromatography Protocols

Excellent PDF on Column Chromatography including detailed protocol and discussion. Univ. Tulane. - [Read Column Chromatography Protocol PDF]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for configuration, column construction, and column packing for a capillary liquid chromatography system. Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns.  A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. - [Read Configuration Column Construction Column Packing for Capillary Liquid Chromatography]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the second step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. This procedure describes the construction of microchromatographic columns, or micro-tips. - [Read Construction of Micro-Tip for Use in IMAC Protocol]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for determination of neopterin and biopterin by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in rat and human plasma, cell extracts and tissue homogenates. - [Read Determination of Neopterin and Biopterin by Liquid Chromatography Protocol]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]

Category: Recombinant Protein Protocols

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography. Biological Procedures Online - [Read Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affin]

Category: Chromatography Protocols: Gel Filtration Chromatography Protocols

Gel Filtration Column Chromatography Protocol. Includes information on: Calibrating the column, Applying standards to the column, Running SDS-PAGE Gels (Tris-Tricine “Schägger” Gels) and includes questions. North Arizona University. - [Read Gel Filtration Column Chromatography]

Category: DNA Protocols: SNP Protocols

High-resolution SNP mapping by denaturing HPLC. A SNP mapping procedure that relies on resolving polymorphisms by denaturing HPLC without the necessity of determining the nature of the SNPs. They demonstrate the use of denaturing high-performance liquid chromatography to identify mutations in the candidate genes and to fine-map chromosomal breakpoints. - [Read High-resolution SNP mapping by denaturing HPLC]

Category: Chromatography Protocols

His Tag Nickel Affinity Chromatography Protocol PDF. The Wallert and Provost Lab. Theory and Introduction: Ni-Affinity Chromatography uses the ability of His to bind nickel. Six histadine amino acids at the end of a protein (either N or C terminus) is known as a 6X His tag. Nickel is bound to an agarose bead by chelation using nitroloacetic acid (NTA) beads. Several companies produce these beads as His Tagged proteins are some of the most used affinity tags in today’s market. - [Read His Tag Nickel Affinity Chromatography Protocol PDF]

Category: HPLC Protocols

HPLC Protocol. A general HPLC procedure to get you started on High Performance Liquid Chromatography. - [Read HPLC Protocol]

Category: RNA Protocols: mRNA Extraction Protocols

Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography. Total RNA is first isolated from the tissues or cells and then mRNA is isolated by PolyA+ selection using oligo(dT) cellulose. This is necessary for all tissue sources rich in RNase (and cell lines). Lazo Lab - [Read Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Isolation of poly A+ RNA from Total RNA by oligo(deoxythymidine)cellulose Chromatography Protocol. Faulkner-Jones - [Read Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Two methods are provided for purifying glycoproteins using wheat-germ agglutinin or concanavalin A-Sepharose.  Because lectin-affinity matrices can bind a few milligrams of protein per milliliter of affinity matrix, only a small amount of affinity gel matrix is required.  The batchwise method is recommended when protein volume is large. - [Read Lectin-Agarose Affinity Chromatography Protocol]