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10 Nov 2019
Assume that you are instead in purifying b-galactosidase from the rest of the proteins found in your cell extract. It is very useful in protein purification to know how much you have purified the protein of interest from other proteins. Protein purification is a multi-step process, and each step corresponds to a specific purification technique that removes a subset of the contaminating proteins while preserving the protein of interest. Usually, the purification starts with a precipitation step where some contaminants precipitates while the protein of interest and a fraction of the contaminating proteins remain in the supernatant. This supernatant is then submitted to different type of chromatography that separate proteins based on their properties: charge, hydrophobicity or size. As the purity of the protein fractions increases, the fractions will have less protein (because of the purification) and less total activity (unavoidable loss of the protein of interest). However, the fractions will be enriched in the protein of interest and the specific activity of the protein should increase. The following table contains information regarding the purification of beta-galactosidase. The yield for step N is defined by: (Total activity in step N/Total activity in step 1) The purification (X-fold) for step N is defined by: (Specific activity in step N/specific activity in step 1) Complete the table (see above). What type of experiment would you propose to determine the purity of the beta-galactosidase fraction obtained after step 4? During a fifth purification step, the total amount of protein decreases and the total activity increased. How would you explain this observation?
Assume that you are instead in purifying b-galactosidase from the rest of the proteins found in your cell extract. It is very useful in protein purification to know how much you have purified the protein of interest from other proteins. Protein purification is a multi-step process, and each step corresponds to a specific purification technique that removes a subset of the contaminating proteins while preserving the protein of interest. Usually, the purification starts with a precipitation step where some contaminants precipitates while the protein of interest and a fraction of the contaminating proteins remain in the supernatant. This supernatant is then submitted to different type of chromatography that separate proteins based on their properties: charge, hydrophobicity or size. As the purity of the protein fractions increases, the fractions will have less protein (because of the purification) and less total activity (unavoidable loss of the protein of interest). However, the fractions will be enriched in the protein of interest and the specific activity of the protein should increase. The following table contains information regarding the purification of beta-galactosidase. The yield for step N is defined by: (Total activity in step N/Total activity in step 1) The purification (X-fold) for step N is defined by: (Specific activity in step N/specific activity in step 1) Complete the table (see above). What type of experiment would you propose to determine the purity of the beta-galactosidase fraction obtained after step 4? During a fifth purification step, the total amount of protein decreases and the total activity increased. How would you explain this observation?
Nestor RutherfordLv2
16 Jul 2019