Posted: February 17th, 2022

Chromatography for Protein Purification

Division of Chemical & Biomolecular Engineering THE NATIONAL UNIVERSITY of SINGAPORE Chemical Engineering Course of Laboratory II Experiment B2 Chromatography for Protein Purification Title Matric No. Group : : : Date of Expt. : GRADE : A. Studying goals 1. 2. three. four. Set up chromatographic assay to find out protein concentrations in a mix. Respect the significance of decision in protein chromatography. Perceive the stress between purity and yield in protein chromatography. Perceive the significance of mass stability closure in protein purification.
B. Introduction I. Quick Protein Liquid Chromatography (FPLC) Excessive Efficiency Liquid Chromatography (HPLC) is the workhorse for any biopharmaceutical protein downstream processing prepare, that includes no less than twice inside the prepare. You will need to recall experiencing the HPLC in one of many experiments in your CN2108 module. Learn up on the important components of the HPLC system. On this experiment, you’ll use a modification of the HPLC, the FPLC (Quick Protein Liquid Chromatography System) to separate and purify a mix of two proteins.
The FPLC has been developed to particularly benefit from the decision functionality of the HPLC for protein purification and assortment. II. Ideas in LC When a mix of proteins is injected into an LC column, the proteins work together with the stationary section based mostly on their respective chemistries and transfer by the column at totally different pace. Primarily based on this differential migration, the proteins elute from the top of the column at totally different occasions and due to this fact develop into separated. This course of is often facilitated by following the proteins with a cellular section.

Though the protein combination could have entered as a slim, concentrated peak, it should exit dispersed and diluted by the cellular section. That is referred to as bandspreading. Bandspreading (which is an inverse indication of the column effectivity) have to be minimized particularly for large-scale protein purification. When bandspreading is extreme, the proteins is probably not sufficiently resolved inside an affordable timeframe. The diploma of separation of 1 part from one other is known as the decision (RS), decided based mostly on equation 1 (discuss with Fig. 1): RS = VB ? V A zero. (W A + WB ) …Eqn. (1) Injection wA VA VB wB Determine 1. Typical protein chromatogram Be aware that decision can be outlined based mostly on retention occasions, as a substitute of volumes. There are numerous methods to enhance decision, probably the most easy of which is to differ the mode of elution – isocratic versus gradient. Each of those modes are based mostly on the power of the cellular section, which instantly impacts the interactions between the proteins and the stationary section. In protein chromatography, it’s fascinating to have excessive yield in addition to excessive purity of the collected fraction.
Yield is the quantity of a protein collected as a fraction of the overall quantity of the identical protein fed, whereas purity is a measure of how a lot of that protein is within the fraction collected. C. Experimental I. Protein Quantification You’ll design an experiment to acquire the calibration curves for the 2 proteins supplied utilizing FPLC. You might be supplied with the next for this experiment: 1. An FPLC system which has been correctly arrange and equilibrated. You solely must inject 100 µL of every of your samples, and your knowledge will probably be recorded and analysed by the pc.
Be aware the profile of the cellular section programmed. 2. A protein combination containing two proteins (S1 and S2) at concentrations of 1. zero mg/mL every. II. Protein Purification and Assortment You might be to carry out a chromatographic purification of 1mL of the protein combination supplied. You may count on the chromatogram proven in Fig. 2. Myoglobin Lysozyme Determine 2. Chromatogram of two proteins from FPLC Primarily based on Determine 2, you might be to conduct the next: 1. Accumulate one fraction of the very best yield that’s 100% pure S1, and the stability in one other fraction. 2.
Accumulate one fraction containing as a lot of S1 fed as doable. D. Dialogue 1. 2. Briefly describe the experiment that you just designed in CI. Clarify your selection of the gathering occasions for every of your assortment in experiment CII. Decide the yield and purity of every of your collected fractions. Carry out a cloth closure for every of CII (1) and CII (2). Primarily based in your leads to (2) above, rationalize the significance of decision in chromatographic separations. Given the stress between yield and purity, which, in your opinion, is extra essential, yield or purity?
How do you intend to enhance the decision of S1 and S2 on this chromatographic purification? three. four. Helpful Notes 1. Reagents: a. Combination of two proteins b. Cellular section – 2M ammonium sulfate in 100mM Phosphate buffer pH 7. zero c. Elution – 100mM Phosphate buffer pH 7. zero FPLC to be arrange with the suitable parameters: a. detection wavelength at 280nm b. pattern loop – 100 µL c. HIC column for protein separation. d. cellular section – 2 M ammonium sulfate e. cellular section move price: 1 mL/min f. gradient elution – linear gradient 100% to zero% over 10 column quantity. 2.

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