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Protein Concentration & Buffer Exchange

Protein concentration using porous membrane filters. Excess liquid fluid passes through the membrane while large protein molecules are retained

Protein concentration and buffer exchange are commonly used in protein and antibody purification, bioprocessing, and proteomic analysis workflows. Sample preparations of macromolecular solutions such as proteins, enzymes, antibodies, and viruses often yield large volumes of diluted proteins and other biomolecules in buffers that are incompatible with downstream processes or detection. Following sample extraction, dilute proteins often need to be concentrated and clarified prior to analysis or use.

Protein Concentration

Several methods exist for protein concentration, including filtration with porous membrane filters, dialysis in hyperosmotic solutions, and precipitation/salting out. Ultrafiltration provides a convenient method that utilizes semi-permeable membranes to concentrate proteins under pressure or centrifugal force with minimal denaturation, deactivation, and degradation of the proteins. Using this technique, excess fluid passes through the filter while target protein is retained, increasing the protein concentration by decreasing the sample volume.

Protein Enrichment

In protein enrichment, specific groups or types of proteins in a biological sample are concentrated for further analysis. Protein enrichment methods are used to isolate low-abundance proteins and reduce sample complexity. Enrichment separates and concentrates target proteins for improved downstream proteomic analysis. Ultrafiltration is often used in protein enrichment to separate classes of proteins based on molecular weight.

Buffer Exchange and Desalting

Purified proteins often need to be transferred to a suitable buffer for further analysis. Buffer exchange, desalting, and detergent removal can be accomplished using methods including:

  • Dialysis: Small permeable molecules such as salts, detergents, solvents, and other impurities are removed based on their ability to pass through a membrane.
  • Column chromatography: Salts are removed using gel filtration columns that rely on size exclusion for separation.
  • Diafiltration: Salts, detergents, and other impurities are removed based on their molecular size using porous ultrafiltration membranes under pressure or centrifugal force.

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