Downstream processing. After completing the purification of the enzyme as descri
ID: 143275 • Letter: D
Question
Downstream processing. After completing the purification of the enzyme as described in question 4, the fractions containing the protein are removed from the fraction collector on the FPLC instrument and pooled, and the volume is then measured to be 3 mL. What is the concentration of the protein? The mobile phase used during size exclusion chromatography was composed of 50 mM sodium phosphate (pH 8) and 150 mM NaCl. However, it just so happens that phosphate ions will interfere with the next experiment that is planned for the protein, and you unfortunately forgot about this when setting up the FPLC instrument. But not all is lost - you can dialyze away the phosphate and replace it with Tris-HCl buffer, which won't interfere. What will be the final concentration of phosphate if you dialyze the protein twice, successively, against 500 mL of a buffer containing 50 mM Tris-HCl (pH 8) and 150 mM NaC1? (You can assume that the dialysis tube reaches equilibrium with the surrounding buffer each time before the buffer is exchanged.) [Please show all relevant computational work.] a. b.Explanation / Answer
Answer d or 4
Size exclusion chromatography separates proteins on the basis of differences in their molecular size. This is suitable technique for polishing steps in purification while sample volumes have been reduced . Samle volumes affects resolution in gel filtration chromatography . Buffer conditions are changed to suit the sample protein or the requirement for further purification, analysis or storage. Purified proteins are collected in the selected buffer.
Size exclusion chromatography separates larger proteins from smaller ones as large molecules travel faster through the cross lynked polymer of the chromatographic column and do not fit into the pores of the polymer while smaller proteins do and take longer time to travel through the chromatographic column.
There are other methods to determine the purity of protein enzyme-
Composition based and Activity based analyses
Electrophoretic method
Sedimentation velocity methods
Mass spectrometry methods
Light scattering methods
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