the image is the steps for help. Not writing assignment!!!!What is the intended outcome of the lysozyme-based ion exchange chromatography described above? Will the protein be effectively separated using the materials listed in the image, or will it fail? Give a brief explanation for your decision. Chromatography is used to separate organic compounds on the basis of their charge, size, shape, and theirsolubility's. A chromatography consists of a mobile phase (solvent and the molecules to be separated) and astationary phase either of paper (in paper chromatography) or glass beads, called resin (in column chromatography),through which the mobile phase travels. Molecules travel through the stationary phase at different rates because oftheir chemistry.In ion exchange chromatography, the glass beads of the column have a charge on them (either + or -). A mixture ofproteins is added to the column and everything passes through except the protein of interest. This is because thecharge of the beads is picked to have the opposite charge of the protein of interest. If the charge on the bead ispositive, it will bind negatively charged molecules. This technique is called anion exchange. If the beads arenegatively charged, they bind positively charged molecules (cation exchange). Thus, a scientist picks the resin to usebased on the properties of the protein of interest. During the chromatography, the protein binds to the oppositelycharged beads.In order to collect the molecules that are bound to the column, they must first be released from the beads in thecolumn. This is called elution. It can be done in one of two ways:1. Changing the charge of the molecules in the sample by running a buffer with a different pH through thecolumn. This affects the shape of the bound molecule and changes the overall charge.2. Displacing the bound molecules with something else, e.g. ions that are more attracted to the beads. Theywill displace the molecules already bound. Using a pipette, add 2 mL of suspended DEAE SEPHAROSE (anion) resin to a chromatographic column.Repeat the same step to another column. Make sure the column valve is closed. Let it settle by gravityuntil you see a defined and settled top.2. Open the column valve and let the preservative liquid slowly drip through until there remains only tinybit on top of the column. Close the column valve. Repeat the same step to another column.3. Add 10 mL of equilibration buffer and let it pass through the column (to wash out preservative).Discardas waste. Repeat the same step to another column. Close the valve when 8 mL of the equilibration bufferpass through the column.4. Label 2 sets of test tubes 1-6. Label one set for lysozyme and the other set for albumin.5. Using a micropipette, gently add 200 µL of lysozyme solution to the top of the first column (withoutdisturbing the top of the bed). Open the column valve and allow the sample to "load" into the column.Collect the flow through into tube #1 for the first protein set. Close the column valve before the columnruns dry. Repeat the same procedure on the second column by adding 200µL of albumin solution.6. Add another 2 mL of equilibration buffer and let it pass through the column slowly. Collect the flowthrough into test tube # 2.7. Repeat step 6, three more times for the column, collecting fractions slowly into test tubes #3, 4, 5respectively.8. Add 2 mL of elution (high salt) buffer to the top of the column. Open the valve.9. As the elution slowly comes through, collect the final 2 mL sample in test tube #6. Close the columnvalve just before the column runs dry.10. Test each fraction in your "run" with Bradford reagent to determine the presence of protein, byfollowing the directions below. Blue color indicates the presence of protein.a) Turn on the spectrophotometer. Let it warm up 15 minutes.b) Add 3.0 mL of Bradford reagent to each of the test tubes 1-6 for each of the two protein sets.c) Cover each of the tubes with a tiny square of Parafilm.d) Invert each tube 3x to mix. Be gentle. Don't allow the solution to foam up. Don't mix up the Parafilmcovers. Wait for 2 minutes before taking absorbance readings.e) Transfer the solutions to proper spec cuvettes (plastic)f) Make up a blank with 2.0 mL of equilibration buffer and 3.0 mL of Bradford reagent. Mix.g) Set the Spec to 595 nm. Collect the baseline using the blank following the instructions for thespectrophotometer.h) Wipe the outside of each spec tube and read the absorbance of each set of samples 1-6

Lysozyme based ion exchange chromatography= Lysozyme is a enzyme which is formed in body naturally…

Answered: the image is the steps for help. Not…

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...