Determination of Protein Concentration Experiment

Determination of Protein dousing ExperimentABSTRACTThe objective of this examine is to determine the preoccupancys of RNase H which was purified in the front science lab test and of an strange solution, which was administered by the TA. A Bradford reagent was utilized to determine the intact ducking which binds to the protein. The try ons were placed in a spectrophotometer and the absorbance was recorded for each sample. The info was compared to the standardisation curve made exploitation the received protein solutions and the absorbance rendering. Our sample was unknown 3, which had a total concentration of 1.418 mg/mL. The concentrations for the maestro pass by means of, washing pilot film fall through, and the eluting buffer storage decrease through were 0.021 mg/mL, 0.0274 mg/mL, and 0.014 mg/mL, respectively, with a 98% confidence interval of 0.0021 %.INTRODUCTIONIn the previous lab prove, the His-tag protein RNase H was purified by implementing a metho d called analogy chromatography. Affinity chromatography is utilized to isolate and purify proteins due to its high selectivity to the protein of pastime (Biochemistry, 2015). The impudent eluting buffer, original flow through, eluting buffer flow through, and the washing buffer flow through were all contain and stored for the latter experiment 7. In addition to the solutions mentioned, five standards protein solutions are hustling by diluting a 1.56 mg/mL of bovine gamma globulin solution (immunoglobulin G) and un utilise eluting buffer. The final wide-awake solution is placed in a spectrophotometer and set at an absorbance of 596 nm. The unused eluting buffer is used as the hold in the experiment to balance the spectrophotometer.A calibration curve is created victimization the absorbance measured from the five standards. In most cases, the calibration curve or standard curves are generated using a least deuce sets of data or replicates, which holds true in our case. Howeve r, the average of the two absorbance sets were used to create the standard curve. The blank/control consists of a buffer without addition of protein. The protein standards have a known concentration of protein, and the unknown sample is the solution to be assayed (Lab Manual). All of the 20 prepared solutions contain the Bradford protein assay.Bovine gamma globulin is a protein assay that is used as a protein concentration reference standard for use in the Bradford for total protein assay. It is used in Bradford and other protein assays. The Bradford protein assay, also referred to as Bradford reagent, is commonly used in laboratories to determine the concentration of the protein within the sample. The reagent binds to the proteins bear witness. The amount of protein present is proportional the binding of the Bradford reagent. Meaning that the more protein present in the sample, the greater the dyestuff will bind. The reagent is said to colorimetric, on that pointfore, a color cha nge can be observed, in reference to the protein concentration. The glowering hue from the reagent becomes progressively darker as we add the protein concentration. The opposite holds true for the lighter blue hue, which indicates less protein is present in the solution. With the aid of the Bradford Reagent, it is possible to determine the total amount of protein concentration present in the sample of interest.EXPERIMENTAL PROCEDURESMATERIALSUnused Eluting cushion (Control)-from lab 6Eluting pilot film flow through (EB)-from lab 6Washing Buffer flow through (WB)-from lab 6professional fertilize through (OFT)-from lab 61 mL of a 1.56 mg/mL Solution of Bovine Gamma Globulin (IgG)0.25, 0.50, 0.75, 1.0, and 1.5 mg/mL of IgG-Protein Concentration StandardsBradford Reagent20 plastic test tubesCuvets, disposable plasticParafilmSpectrophotometerPROCEDURESPrepare five standard protein solutions in a microcentrifuge tube by diluting the 1.56 mg/mL of IgG stock solution appropriately with unused eluting buffer from lab 6. individually solution should have a total volume of 250 L and the concentrations should be as follows 0.25, 0.5, 0.75, 1.0, and 1.5 of IgG. band up 20 test tubes and divide into two sets. Label 5 test tubes per the concentrations mentioned above, and the remaining 5 tubes as follows eluting buffer flow through, unused eluting buffer, original flow through, washing flow through, and the unknown sample obtained from the TA. Repeat this procedure for set two. Add 50 L of the appropriate solution to each tube. Add 1.5 mL of Bradford reagent to each of the 20 test tubes then cover with each tube with parafilm and mix using the vortex mixer for approx..3 seconds. Wait 10 minutes and then channel all the solutions to cuvets. For set one and two, place the unused eluting buffer cuvet into the spectrophotometer setup to balance and set to 596 nm. Read the absorbance for the other cuvets. Record each reading in lab notebook. Plot the average absorbance on the y-axis vs. concentration on the x-axis of the duplicate standard protein solutions. Determine the concentrations of the original flow through, washing flow through, eluting buffer flow through, and the unknown sample by using comparability generated from the slope of the plot.RESULTSDATA TABLES Standard Protein SolutionsProtein Concentration (mg/mL)Volume of 1.56 mg/mL IgG Stock Solution Needed (L)Volume of Unused Eluting Buffer Used (L)Total Volume of Solution0.2540210250 l0.5080170250 l0.75120130250 l1.016090250 l1.524010250 lAbsorbance DataConcentrations (mg/mL)Absorbance at 596 nmSet 1Set 2Average1.500.6080.6330.6211.000.4550.4230.4390.750.4410.2460.3440.500.1620.3070.2350.250.0430.0560.050Unused EB (Control) = 00.0000.0000.000 current hunt through0.004-0.0030.001Washing Buffer0.0020.0050.004Eluting Buffer0.001-0.004-0.002Unknown sample 30.6080.6060.607Total ConcentrationProtein Sample Concentration (mg/mL)Original Flow through0.0205Washing Buffer0.0274Eluting Buffer0. 0136Unknown Sample 31.418GRAPHCALCULATIONSThe expression given by the best flout elongated trend will be used to determine the concentrations for the original flow through, washing flow through, eluting buffer flow through, and unknown sample 3. Where y-represents the absorbance, and x-represents the concentration. The units like to the slope are in mg/mL. Equation of the slope is y = 0.4336x 0.0079. Solving for x will give us the concentrations of the samples.Concentration of the Original Flow ThroughRearrange to brighten for xy = 0.4336x 0.0079 x = (y 0.0079)/(0.4336)(0.001) = 0.4336x 0.0079x = (0.001 + 0.0079)/(0.4336)x = 0.0205 mg/mL 0.021 mg/mLCALCULATIONS (continued)Concentration of Washing Buffer Flow Throughy = 0.4336x 0.0079(0.004) = 0.4336x 0.0079x = (0.004 + 0.0079)/(0.4336)x = 0.0274 mg/mL 0.027 mg/mLConcentration of Eluting Buffer Flow Throughy = 0.4336x 0.0079(-0.002) = 0.4336x 0.0079x = (-0.002 + 0.0079)/(0.4336)x = 0.0136 mg/mL 0.014 mg/mLConcentration o f Unknown Solution 3y = 0.4336x 0.0079(0.607) = 0.4336x 0.0079x = (0.607 + 0.0079)/(0.4336)x = 1.418 mg/mL DISCUSSIONThe main goal for this experiment was to determine the protein concentration of the original flow through, washing buffer flow through, eluting buffer flow through, and an unknown sample which was given by the TA. These samples apart(predicate) from the unknown were prepared in the previous lab experiment and was retained for further analyses for this experiment. In addition to the samples mentioned above, five standard protein solutions were prepared by diluting the 1.56 mg/mL of IgG solution with the unused eluting buffer obtained from lab 6. The concentrations of the standards were as follows 0.25 mg/mL 0.50 mg/mL, 0.75 mg/mL, 1.0 mg/mL, and 1.5 mg/mL. The unused eluting buffer was also used as the control. It was apparent that upon adding the Bradford reagent to the samples there was visible a color change. A few of the solutions instantly cancelled to a darke r blue hue while others remained a light blue color. The darker color indicated there was a greater concentration of proteins.A calibration curve was generated by plotting the concentrations of the five standards and their respective absorbance reading. It was determined that the best fit for the data was linear which yields an par in the form of y = mx + b, where y represents the absorbance at 596 nm and x represents the protein concentration. The equation was rearranged as to solve for x and determine the protein concentration of the samples and the unknown 3, based on the data from the averages of the two sets of absorbance measured. However, two values from set two gave negative readings, which was declaratory that the concentration of the protein was less than that of the control sample.As per my results, it was concluded that our unknown sample had a total protein concentration of 1.418 mg/mL. The concentrations for the flow through of the original solution, washing buffer, and eluting buffer had a total protein concentration of 0.0205 mg/mL, 0.0274 mg/mL, and 0.0136 mg/mL, respectively. The absorbance data was further analyzed using a 98% confidence interval and yielded a 0.002% margin error. The R-value was relatively close to 1, which gives an equation that is more accurate. This also indicates that the calculated values for the protein concentration will ring a value closer to the true concentration of the protein of interest.REFERENCESJ. M. Berg, J. L. Tymoczko, G. J. Gatto, Jr., L. Stryer, Biochemistry (8th ed., pp. 70-71). (2015). W.H. Freeman Company.Bradford, M. M. uninflected Biochemistry. Volume 72. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the dominion of Protein-dye Binding. (pp. 248-254). (1976).Robyt, J. F. and White, B. J. Biochemical Techniques. Theory and Practice. Brooks/Cole, Monterey, CA. (1997)https//www.thermofisher.com/order/catalog/product/23212