column Protocols

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

PDF. A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris. CHARLES R. ALLERSON, ALAN MARTINEZ, EMINE YIKILMAZ and TRACEY A. ROUAULT. RNA Journal. - [Read A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol for ABI prism 310 cycle sequencing. Includes: Sequencing Reaction; Column Purification; Sample Loading Preparation. - [Read ABI Prism 310 Cycle Sequencing Protocol]

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: Protein Protocols: Antibody Purification Protocols

ANTIBODY PURIFICATION ON THE PROTEIN G COLUMN. Jorker Lab, Caltech. - [Read ANTIBODY PURIFICATION ON THE PROTEIN G COLUMN]

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: Molecular Biology Protocols: Phage Protocols and Methods: Lambda Phage Protocols

Column Method for Mini-prep of lambda phage DNA purification from lysates. Donis Keller Lab - [Read Column Method for Lambda Phage DNA Preparation]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Mini-prep method for lambda phage DNA purification from lysates. - [Read Column Method for Lambda Phage DNA Preparation Protocol]

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: 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: ELISA Protocols: ELISpot Protocols

Tips for selecting the “right” ELISPOT filter plate and making the best protocol choices. Alan J. Weiss ZellNet - [Read Elispot plates column]

Category: Chromatography Protocols

FPLC Protocol. The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one. Sir William Dunn School of Pathology, Oxford University. - [Read FPLC Protocol]

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: Chromatography Protocols: Liquid Chromatography Protocols

Protocols and information on general HPLC column care. Includes: Silica based columns and Polymer based columns.  Protocols included: pH stability, Mechanical stability, Mobile phases (Eluents), Proper storage of HPLC columns, Equilibration time, Regeneration of a column, Regeneration of RP packings, Regeneration of NP (Normal Phase) packings, Regeneration of Ion Exchange Packings - [Read HPLC Column Care Protocols and Information]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for packing an IMAC column. Protocol describes a procedure for packing a 30-ml immobilized metal-ion affinity chromatography (IMAC) column with the aid of a pump. - [Read Packing an IMAC Column Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the preparation of a prepacked IMAC column. Purification of histidine-tagged proteins on a milligram scale can be performed conveniently on a small (1to 5-ml) immobilized metal-ion chromatography (IMAC) column using a syringe to load the sample, wash the column, and elute the protein. - [Read Preparation of a Prepacked IMAC Column Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the preparation of ion-exchange chromatography column.  Ion-exchange chromatography (IEC) can be used as a crude step in a protein purification scheme, or, with proper preparation, as a high-resolution step.  If high resolution is desired, considerable care should be taken during column preparation, choice of IEC media, and column packing. - [Read Preparation of an Ion-Exchange Column Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: Protein Protocols: Protein Precipitation Procols

Protein Precipitation. Developed by: Eric Burtson. American Association of Immunologists 1995. In this lab you will separate a solution of proteins using protein precipitation. Since you will be testing the same protein mix that you used in the Gel Filtration lesson, you will pass the protein through a gel filtration column to identify which protein(s) precipitates. - [Read Protein Precipitation]

Category: Protein Protocols: Protein Purification and Identification

Protein Purification: Column Chromatography - Ion exchange; Dialysis and Concentration. Blaber lecture - [Read Protein Purification: Column Chromatography - Ion exchange; Dialysis and Concentration]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies.  Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins.  It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins, constructed in pGEX vectors, are fused to glutathione S-transferase (GST) and can be purified to near homogeneity by affinity chromatography on glutathione-agarose.  Bound GST-fusion proteins are readily displaced from the column by elution with buffers containing free glutathione. - [Read Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins engineered to have a polyhistidine tail at either the carboxyl or amino terminus can easily be purified in one step by affinity chromatography on a resin carrying chelated nickel ions.  Chromatography can be carried out in column or batch formats.  After unbound proteins are washed away, the target protein is eluted using imidazole, which typically preserves the antigenic and functional features of the protein. - [Read Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol]