Introduction
Protein A Agarose is prepared by covalently coupling purified Protein A to 4% crosslinked agarose beads by a cyanogen bromide method. Protein A Agarose can bind >18 mg of human IgG per mL of gel, a value comparable to that reported by Hjelm et. al (1).
Protein A Agarose binds specifically to the Fc region of IgG molecules. As a result of this property, Protein A Agarose has been used to accomplish the following:
- Purification of IgG (1,2)
- Fractionation of IgG fragments (2)
- Fractionation of cells (3)
- Isolation of antigen-antibody complex (4)
Contents
Protein A Agarose is supplied as a suspension in buffer containing 0.02 M sodium phosphate, (pH 7.2), 0.15 M sodium chloride, 0.02% (w/v) merthiolate.
Intended Use
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Product Qualification
The Certificate of Analysis provides detailed quality control information for each product. Certificates of Analysis are available on here.
5 Steps to Protein Isolation and Purification
Protein purification is an essential component of protein research. The study of protein function, structure, and interactions heavily relies on the purity and quality of the isolated protein of interest. Here we present to you a five-step workflow that will help you in your quest. Learn about methods and technologies for protein expression, protein extraction and preservation, protein purification, protein clean up, and protein quantitation and detection.
Follow these 5 steps to obtain optimal protein sample conditions
Choosing the right expression system for your production needs
Researchers have many methodology choices when it comes to producing recombinant proteins for early-stage discovery research through large-scale production of biotherapeutic drugs, vaccine development, and structural studies. It is imperative to use the right protein expression system for the target protein and application of interest. We offer a wide selection of superior mammalian, insect, bacterial, and yeast protein expression systems to suit your research needs.
Our Gibco Expi Transient Expression Systems, available in mammalian (CHO-S, 293F cells) and insect (Sf9 cells) expression formats, are completely optimized systems that enable rapid, high-yield production of proteins.
Extract and stabilize your target protein from the sample
Protein extraction techniques vary depending on the source of the starting material, the location of the protein of interest within the cell, and the downstream application. Other important considerations include the preservation of protein activity and function as well as the reduction of background effects.
Protein extraction
Tissue and cell lysis
Historically, mechanical disruption has been used to lyse cells and tissues; our gentle, detergent-based solutions have been developed to efficiently lyse cells and enable the separation of subcellular structures without requiring physical disruption, providing high yields of active proteins.
Protein extraction product features:
- Optimized—formulations maximize protein yield and preserve protein activity
- Efficient—only produces minimal cross-contamination between subcellular fractions
- Compatible—extracts can be used directly in most downstream applications
- Gentle—eliminates the need for mechanical cell disruption for most sample types
High protein yield from a variety of mammalian cell types and cellular compartments
Efficient and selective enrichment of membrane proteins
Detergent solutions
Detergents are frequently used in cell lysis reagent formulation and other protein research methods. Thermo Scientific Surfact-Amps Detergent Solutions are highly purified, precisely diluted (10%) formulations that are ideal for applications or assays that are sensitive to contaminants present in unpurified detergents.
Protein detergent product features:
- Accurate—precise 10% detergent solution in ultrapure water
- Easy-to-use—solution is simple to dispense and dilute
- Exceptionally pure—less than 1.0 μq/mL peroxides and carbonyls
- Stable—packaged under inert nitrogen gas in glass ampules or HDPE bottles
Protein stabilization
Cell lysis disrupts cell membranes and organelles, resulting in unregulated enzymatic activity that can reduce protein yield and function. To prevent these negative effects, protease and phosphatase inhibitors can be added to the lysis reagents. Numerous compounds have been identified that can inactivate or block the activities of proteases and phosphatases.
Protease and phosphatase inhibitor product features:
- Convenient—ready-to-use, fully disclosed, broad-spectrum formulations available as either liquid cocktails, tablets, or capsules, in multiple pack sizes and with a minimum of 1 year of shelf life
- Complete protection—all-in-one formulations containing both protease and phosphatase inhibitors are offered in both liquid and tablet formulations (with EDTA or EDTA-free)
- Compatible—use directly with Thermo Scientific Pierce Cell Lysis Buffers, other commercial, or homemade detergent-based lysis reagents
Broad effective inhibition of proteases and phosphatases
Tips
- Cell lysis disrupts cells membranes and organelles resulting in unregulated proteolytic activity that can reduce protein yield and function. To prevent extracted protein degradation, it is often necessary to add protease and phosphatase inhibitors to cell lysis reagents.
- Most researchers use a mixture of several different inhibitor compounds to ensure the protein extracts do not degrade before analysis of the target of interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
- Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.