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Analysis of Protein Aggregates Molecular Weight by SDS-PAGE (CAT#: STEM-B-0301-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.

Molecular weight, also called molecular mass, mass of a molecule of a substance, based on 12 as the atomic weight of carbon-12. It is calculated in practice by summing the atomic weights of the atoms making up the substance's molecular formula.




Principle

When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins. In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length.

Applications

Biopharmaceutica

Procedure

1. Gel production: When using different buffers in the gel (discontinuous gel electrophoresis), the gels are made up to one day prior to electrophoresis, so that the diffusion does not lead to a mixing of the buffers.
2. Sample preparation: During sample preparation, the sample buffer, and thus SDS, is added in excess to the proteins, and the sample is then heated to 95 °C for five minutes, or alternatively 70 °C for ten minutes.
3. Electrophoresis
4. Gel staining
5. Analysis: Protein staining in the gel creates a documentable banding pattern of the various proteins.
6. Archiving: After protein staining and documentation of the banding pattern, the polyacrylamide gel can be dried for archival storage. Proteins can be extracted from it at a later date.

Materials

• Sample: Proteins
• Equipment: SDS-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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