Analysis of Protein Aggregates Charge and Hydrodynamic Size by Native-PAGE (CAT#: STEM-B-0312-CJ)

Introduction

The generic term 'aggregates' refers to species characterized by a wide size range, diverse morphologies and structures. Protein aggregates may start in the low nanometer size range but then can grow into the micrometer and even visible size range.

Most protein therapeutics and many other biopharmaceutical compounds are inherently unstable and can undergo aggregation through various pathways. Aggregates of various kinds can be formed, such as reversible and non-reversible, soluble, and non-soluble etc. In addition,Aggregation maybe occur because of exposure to air-liquid or liquid-solid interfaces, e.g., during mixing, during filling and shipping, during reconstitution of lyophilized products, or through contact with chromatography columns, pumps, pipes, vessels, filters, etc. Aggregation can directly influence the efficacy of the therapy by reducing the number of functional molecules, but also indirectly influence efficacy as well as safety of a therapy by inducing side-effects, such as unwanted immunogenicity.

The hydrodynamic dimension of a protein is a reflection of both its molecular weight and its tertiary structures. Studying the hydrodynamic dimensions of proteins in solutions can help elucidate the structural properties of proteins.

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Principle Applications Procedure Materials Notes Related Products

Principle

In native PAGE, proteins are separated according to the net charge, size, and shape of their native structure. Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers. And Polyacrylamide Gel Electrophoresis is based on the principle of migration of charged particles under the influence of electric field to separate out proteins and nucleic acids.

Applications

Biopharmaceutica

Procedure

1. Sample preparation: Samples may be any material containing proteins or nucleic acids. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins.
2. Preparing acrylamide gels: The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization.
3. Electrophoresis: Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
4. Further processing: Following electrophoresis, the gel may be stained (for proteins, most commonly with Coomassie brilliant blue R-250 or autoradiography; for nucleic acids, ethidium bromide; or for either, silver stain), allowing visualization of the separated proteins, or processed further (e.g. Western blot).

Materials

• Sample: Purified Proteins
• Equipment: Native-PAGE

Notes

• The formation of aggregates in your biopharmaceutical product can have a negative effect on safety, efficacy and function. Regulatory authorities expect that orthogonal characterization techniques are used to fully understand the aggregation profile of any molecule.
• Measurable range: KDa–MDa

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