Using a detailed scheme, propose a step-wise protocol to purify protein B by ion exchange chromatography (Explain your logic/choices).
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Using a detailed scheme, propose a step-wise protocol to purify protein B by ion exchange chromatography (Explain your logic/choices).
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- Protein Solubility 3.5 5.5 5 5 0.068 0.028 Absorbance 0.098 Conc.(mg/ml) 0.195 0.130 0.044 0.9% 2.786% 9.429% 11.3: % solubility 4.179% The above table indicates the concentration of protein in the diluted supernatant and the supernatant before dilution at different pH. Dilution Factor 30 22.5 15 PH 7.5 1.5 4.5 5 0.027 3 0.042 Protein Solubility Versus pH 6.5 6 4.5 pH Value The above figure shows a plot of protein solubility versus pH. 50 7.5 7.5 50 0.032 0.053 8.5 50 0.054 0.100 21.429% 9 Please provide a brief discussion and explanation of the results. (using isoelectric point and net charge to explain)Figure 3: ● ● ● ● ● ● KDa ● 97.4 66.2 45.0 ● 31.0- 21.5 14.4 S-1 p-1 S-2 2-0 This figure was generated by centrifuging a pura sample of protein, removing the supernatant, and resuspending the pellet in the same volume as the supernatant to allow direct comparison. The supernatant and pellet samples were then prepared for SDS-PAGE identically and run via normal SDS-PAGE procedures. In the figure, "s" means supernatant and "p" means pellet. The text or number after the dash represents a different condition. For example, s-1 and p-1 are the supernatant and pellet samples under condition 1. It is not shown, but under wild-type conditions, essentially all of the protein is found in the supernatant. S-3 ● What does the intensity of each band represent? ● Would you find soluble protein in the supernatant or pellet? Why? Would you find aggregated protein in the supernatant or pellet? Why? For each condition (there are 5 different conditions), is there a higher percentage of the total protein…Given this: Protein Isoelectric pH Molecular weight (kDa) Ovalbumin 4.6 45 Insulin 5.4 5.7 Fibrinogen 5.8 340 Y-globulin 6.6 160 Collagen 6.6 115 Hemoglobin (monomer) 7.1 16 Myoglobin 7.0 16.7 Does all proteins separated properly w/ 2D gel electrophoresis?
- Given this: Protein Isoelectric pH Molecular weight (kDa) Ovalbumin 4.6 45 Insulin 5.4 5.7 Fibrinogen 5.8 340 Y-globulin 6.6 160 Collagen 6.6 115 Hemoglobin (monomer) 7.1 16 Myoglobin 7.0 16.7 Does all proteins separated properly w/ isoelectric focusing?Given this: Protein Isoelectric pH Molecular weight (kDa) Ovalbumin 4.6 45 Insulin 5.4 5.7 Fibrinogen 5.8 340 Y-globulin 6.6 160 Collagen 6.6 115 Hemoglobin (monomer) 7.1 16 Myoglobin 7.0 16.7 What is the appearance after visualization of a protein mixture containing the seven proteins above if isoelectric focusing is done?A protein required 6.8 min to travel 82 cm to the detector in a 96 cm -long capillary tube with 25.4 kV between the ends. Find the apparent electrophoretic mobility. How many femtomoles of 2.4 μM protein are injected electrokinetically into a 50 μm diameter capillary at 5.0 kV for 3.0 sec. if the sample has half of the conductivity of the background electrolyte.
- Protein Solubility 3.5 5 0.028 Absorbance 0.098 Conc.(mg/ml) 0.195 0.044 0.9% 2.786% 9.429% 11.3: % solubility 4.179% The above table indicates the concentration of protein in the diluted supernatant and the supernatant before dilution at different pH. Dilution Factor 30 22.5 pH 15 7.5 1.5 4.5 5 0.027 3 0.042 5.5 5 0.068 0.130 Protein Solubility Versus pH 6 6.5 4.5 pH Value The above figure shows a plot of protein solubility versus pH. 50 Please provide a brief discussion and explanation of the results. 7.5 7.5 50 0.032 0.053 9 8.5 50 0.054 0.100 21.429%The calibration curve shown below was used to analyze an unknown protein solution. What is the concentration of the unknown solution, if the absorbance of the unknown is 0.505? Answer in ug/mL. 1.2 y%3D0.005x+0.061 R=0.992 0.8 0.6 0.4 0.2 50 100 150 200 250 Concentration ug/ml Absorbance2. 0.1 mL of a protein solution of concentration of 7 mg/mL was diluted to a total volume of 4.0 mL with water (i.e. 0.1 mL of the solution was added to 3.9 mL of water). 3 mL of this solution was then mixed with 27 mL of water. What is the concentration of the diluted protein solution? Space to show your workings:
- Protein Isoelectric pH Molecular weight (kDa) Ovalbumin 4.6 45 Insulin 5.4 5.7 Fibrinogen 5.8 340 Y-globulin 6.6 160 Collagen Hemoglobin (monomer) 6.6 115 7.1 16 Myoglobin 7.0 16.7 From the given table, draw the appearance after visualization of a protein mixture containing the seven proteins in the table when subjected to SDS-PAGE.5/ n SO a- 6.46 The following data show the binding of Mg2+ ions with a protein containing n equivalent sites: [Mg2+ ] total/μM [Mg2+ ]free/μM 108 Com 35 180 65 288 501 115 248 752 446 Apply the Scatchard plot to determine n and Ka. The protein concentration is 98 µM.Does this calculation look correct? My goal isto have a target mass of 10 µg of my protein with a total volume of 30 µl. Protein was measured and found to have a concentration of at 308.35 µg/ml Recall, mass = concentration * volume 10 µg = 308.35 µg/ml * V V = 0.01 mg/ 0.30835 mg/mL = 0.032430679 mL = 32.43067942 µL Note: cannot measure this amount; two dilutions required. Dilute by 1/40: measure 2.5 µl of protein at 0.30835 µg/µl and add it to 97.5 diluent to prepare a solution of protein at 7.71 mg/ml. This diluted sample will be used further. Mass = concentration * volume 10 µg = 7.71 mg/ml * V V = 0.01 mg/ 7.71 mg/ml = 1.3 µL Therefore, will measure 1.3 µl of protein at 7.71 mg/ml and add it to 28.7 µl of diluent.