The protein pellets are dissolved in buffer S1 for minutes under continuous agitation with the above vortex-genie 2 model G as shown in Scheme 1. For human urine, after the protein precipitation step, the pellets are washed with deionized water, before addition of the buffer solution prior to solubilization.
A critical step in the process requires that the volume of buffer S1 added to the pellet must be equal to the initial volume of protein added to the microcentrifuge tube to ensure the same treatment, and to accurately estimate the amount of proteins precipitated.
This is shown in equation Three hundred and fifty microliters from the remaining protein solution dissolved in the above buffer buffer S1 is mixed with 0. A volume of 0. Optimum concentration of TCA using central composite design: Central composite design CCD , with five coded levels Table 1 , is used to elucidate the true optimal concentration of the TCA required for protein precipitation.
The 13 experiments represent a CCD with 22 full factorial designs, four axial points, and a center point with 4 replications Table 1. The mathematical model that derives from such a CCD is expressed as the following second-order polynomial equation:.
Table 1: Independent variables and their level in central composite design and dependent variables. Two independent variables Table 1 are chosen based on preliminary screening studies, and the level of the two factors are chosen based on a steepest descent ascent method [ 29 , 30 ]. Table 1 shows the independent variables in physical units, with their associated coded values as well as the dependent variables.
The second order model can be written in matrix notation as follows [ 33 ]:. Briefly 0. Next 0. The final concentration of TNF is 0. Finally the protein is precipitated as described above and the absorbance of the supernatant is recorded by optical scanning between nm. The absorbances of the blanks are 0. The statistical analysis was performed using JMP?
Polynomial equations of the response values of absorbance of the supernatant at nm Y 1 , and the percentage of precipitated protein Y 2 , are derived from the total result of 13 runs in the above CCD design. Experimental variables that significantly affect these responses are identified through a Pareto chart. A theoretical optimum condition is obtained by setting the maximum desirability of maximum protein precipitation yield. A student t-test is used for the checkpoint analysis, and a P-value below 0.
Table 2: Central composite design showing independent variables with measured responses. Where Y 1 is the absorbance of the supernatant; Y 2 is the percentage of protein precipitated; and X 1 and X 2 are the coded independent variables. Based on the above Equations 4 and 5, the vector b and the matrix B values are shown below:. Table 3 shows the ANOVA results to check the significance of the model parameters for both mathematical models that derived from the experimental design.
Table 3: Results of ANOVA analysis for the statistical model parameters for the absorbance of the supernatant Y 1 , and the percentage of the protein precipitated Y 2. Table 4 shows the lack-of-fit test to check the mathematical models adequacy. In other words, this test allows assessment if both equation models for Y 1 and Y 2 can adequately predict the absorbance of the supernatant and the percentage of protein precipitated, respectively.
Table 4: Lack-of-fit test analysis to check the model adequacy predicting the absorbance of the supernatant Y 1 , and the percentage of protein precipitated Y 2. The lack-of-fit test also shows that the model Y 2 adequately fit the data and can predict the percentage of protein precipitated Table 4. The protein absorbency recorded at nm is reproducible, but the model Y 1 cannot be used to adequately predict the final absorbance of the supernatant based on the lack-of-fit test [ 34 ] Table 4.
Moreover, the response surface and the contour plots, which derived from the CCD, are used to characterize the shape of the surface and can locate the optimum using computer software [ 33 ] Figure 1. Figure 1: Three-dimensional responses surface A1 , and contour plot A2 showing the supernatant absorbance data A and those of the percentage of protein precipitated B1 and B2 as a function of volume of protein solution and volume of TCA solution.
The intersections of the two orthogonal lines, in figures A2 , and B2 are the saddle point. The second order statistical model Y 2 is checked in triplicate with two random points with respective X 1 , X 2 values of Bias for the fitted model Y 2 is computed using the following equation Equation 9 :.
Table 5: Checkpoints experiments comparing measured and predicted percentage of protein precipitated. The predicted percentage of protein precipitated Y 2 , and the measured Y 2 values, are statistically insignificant if the p-value p is greater than 0.
Thus, the model Y 2 can accurately predict the Y2 values for a given volume of aqueous solution of BSA solution, and a given the volume of TCA solution 6. Figure 2: Pareto chart showing the effect of the independent variables X1, volume of protein solution; X2, volume of TCA solution , on the absorbance of the supernatant recorded at nm and the percentage of protein precipitated using BCA assay.
Sorted parameter estimates and their corresponding t-ratio are shown on the horizontal-axis. Figure 3: Prediction profiler and desirability showing the effect of the volume of protein solution and the volume of TCA solution on the absorbance of the supernatant measured at nm and the percentage of protein precipitated using BCA assay. In Protein Folding Creighton, T.
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BQC has been recognized during these years as a safe value by organizations, companies and investors. Principals of various protein precipitation methods. Home » Principals of various protein precipitation methods. Principals of various protein precipitation methods View Larger Image.
Protein precipitation kits Acid precipitation This method relies on the changes of a solution pH. Salting precipitation Adding the salt ions into the solution cause the restriction of the available water molecules for the proteins, which leads to destruction of the hydrogen bonds. Alcohol precipitation Organic solvents can be also used.
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