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Analysis of Quaternary Structure by Electron Microscope (CAT#: STEM-B-0381-CJ)

Introduction

Structure and conformation of a biological molecule is key for its function. The higher order structure of a biopharmaceutical molecule is, thereby, often directly connected to the quality, stability, safety, and efficacy of a therapy. The higher order structure is considered a critical quality attribute and, thus, a detailed understanding of the higher order structure of a biopharmaceutical compound is critical in every research and development phase. Characterizing the secondary, tertiary and, if present, quaternary structure of a biopharmaceutical compound requires multiple analytical techniques.<br /><br />Many proteins are made up of more than one polypeptide chain to perform their function. The complete structure of such a protein is designated its quaternary structure, and each polypeptide chain is referred to as a subunit. The quaternary structure is stabilized by the same bonds as for the tertiary structure, including different noncovalent bonds and disulfide bonds. These bonds hold the subunits together and arrange themselves to form a larger protein complex.




Principle

The electron microscope uses a beam of electrons and their wave-like characteristics to magnify an object's image, unlike the optical microscope that uses visible light to magnify images. Electron Microscopes (EMs) function like their optical counterparts except that they use a focused beam of electrons instead of photons to "image" the specimen and gain information as to its structure and composition.

Applications

Biopharmaceutica

Procedure

1. Sample preparation.
2. A stream of high voltage electrons (usually 5-100 KeV) is formed by the Electron Source (usually a heated tungsten or field emission filament) and accelerated in a vacuum toward the specimen using a positive electrical potential.
3. This stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam.
4. This beam is focused onto the sample using a magnetic lens.
5. Interactions occur inside the irradiated sample, affecting the electron beam.
These interactions and effects are detected and transformed into an image.

Materials

• Sample: Protein monomers, Fragments and Aggregates
• Equipment: Electron Microscopes

Notes

• TEM: magnifies 50 to ~50 million times; the specimen appears flat.
• SEM: magnifies 5 to ~ 500,000 times; sharp images of surface features.
• STEM: magnifies 5 to ~50 million times; the specimen appears flat.