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Question: 1 using the passage below explain why was cation exchange...

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1. Using the passage below explain: Why was cation exchange used, and why was a salt solution used to elute column?

An antibacterial protein was isolated from a shore crab species. Eur J. Biochem. 264, 350-357 (1999) FEBS 1999 Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas Juliet M. Relf1, June R. S. Chisholm1, Graham D. Kemp2 and Valerie J. Smith1 Environmental&Evolutionary Biology, Gatty Marine Laboratory Unive rsity of St And rews, Fife, Scotland: Biomedical Sciences, North Haugh, University of St Andrews. Fife. Scotland Extracts of the granular haemocytes of Cacis maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram- positive bacteria. In addition its activity is stable after heating to 100 ℃ and is retained at concentrations as low as 10 μ g·mL-. II has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four- disulphide core proteins. Partial cDNA sequence data has been obtained. Keywords: antibacterial protein; Carcinus; crustacean; haemocytes; innate immunity The first step in the purification of this protein utilizes cation exchange chromatography. The procedure is shown below. Briefly explain why cation exchange is a good choice for this protein and why a salt (NaCI) gradient solution is used to elute or release the protein from the column. Purification The dialysed acid cxtract of granular cells (2 mL). with a protein conten of 800-900 ug mL. was applied to an 8 x 1 em CM Sepharose cation-exchange column equilibrated with 50 mM sodium phosphate (pH 6.5). Unbound material was eluted by extensive washing with buffer at a flow rate of 1mL-min Bound material was eluted with a two-step gradient of 50 mM sodium phosphate (pH 6.5) comaining 0-1 M NaCl as follows: 0-50% NaCI over 30 min and 50-100% NaCl over 15 min. Fractions (1 mL) were collected and absorbances read at 280 nm

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