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Analysis of Protein Aggregates Size and Morphology by Optical Microscopy (CAT#: STEM-B-0298-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.<br /><br />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.<br /><br />The sizes of proteins are relevant to their biochemical structure and for their biological function. The statistical distribution of protein lengths across a diverse set of taxa can provide hints about the evolution of proteomes.




Principle

An optical microscope creates a magnified image of an object specimen with an objective lens and magnifies the image further more with an eyepiece to allow the user to observe it by the naked eye. Put your naked eye in the eye (pupil) position on the eyepiece barrel to observe the enlarged image. And the last image to be observed is an inverted virtual image.

Applications

Biopharmaceutica

Procedure

1. Sample Preparation
2. Preparing the microscope slide with the sample to be examined, including a coverslip over the sample.
3. Adjusting the objective lens to the lowest power of resolution.
Placing the microscope slide with the sample to be examined onto the stage and fasten in place.
4. Adjusting the height of the stage so that it is as close as possible to the objective without contact between the lens and the sample.
5. Viewing the sample through the eyepiece and adjusting the focus knob to bring the image into focus.
6. Changing the condenser and intensity of light to increase the contrast of the image.
7. Moving the microscope slide around to being the part of interest of the sample into the center of the field of view.
7. Adjusting the focus knob, condenser and light intensity once again to improve the clarity of the image.
8. Changing to the next objective lens and readjusting the focus, condenser and light as needed to view the image clearly.

Materials

• Sample: Proteins
• Equipment: Optical Microscopy

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: 1 µm–mm