Extraction and Clarification Procedures

Extraction procedures should be selected according to the source of the protein, such as bacterial, plant or mammalian, intracellular or extracellular. 

Use procedures which are as gentle as possible since disruption of cells or tissues leads to the release of proteolytic enzymes and general acidification. Selection of an extraction technique is dependent as much upon the equipment available and scale of operation as on the type of sample. Examples of common extraction processes are shown in Table 8. Extraction should be performed quickly, at sub-ambient temperatures, in the presence of a suitable buffer to maintain pH and ionic strength and stabilise the sample. Samples should be clear and free from particles before beginning a chromatographic separation. 

Extraction process Typical conditions Protein source Comment 

Gentle Cell lysis (osmotic shock) 2 volumes water to 1 volume packed pre-washed cells erythrocytes E.coli periplasm intracellular proteins lower product yield but reduced protease release 

Enzymatic digestion lysozyme 0.2 mg/ml, 37 °C, 15 mins. bacteria intracellular proteins lab scale only, often combined with mechanical disruption 

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The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure. 

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