4. For routine use, the biuret procedure is simple to perform, producing a stable color that obeys Beer's Law. UV-Vis Spectroscopy is primarily used for quantitative analysis in chemistry and one of its many applications is in protein assays.

5. Hydrated copper sulphate: this provides the cu(II) ions which form the chelate complex. Cu(II) ions give the reagent its characteristic blue color. Potassium hydroxide does not participate in the reaction but provides the alkaline medium. Potassium sodium tartarate: (KNAC4H4O6.4H2O) stabilizes the chelate complex, prevent precipitation of copper hydroxide and potassium prevent auto reduction of copper.

6. One commonly used method for determining the total protein in a sample is the Biuret method. The Biuret method is based on the complexation of Cu2+ to functional groups in the protein’s peptide bonds. The formation of a Cu2+protein complex requires two peptide bonds and produces a violet-colored chelate product which is measured by absorption spectroscopy at 540 nm. Over a given concentration range, the measured absorption at 540 nm is linear with respect to the concentration of total protein.

7. The intensity of the color and hence the absorption at 540nm, is directly proportional to the protein concentration, according to the beer lamber law. Molecules containing 2 or more peptide bonds associate with the cupric ions to form a coordination complex that imparts a purple color to the solution with Amax = 540 nm. The purple color of the complex can be measured independently of the blue color of the reagent itself with a spectrophotometer or colorimeter.

8. Under alkaline conditions cupric ions chelate with the peptide bonds resulting in reduction of cupric ions to cuprous ions. The cuprous ions can also be detected with folin ciocalteu reagent (phosphomolybdic/phosphotungstic acid), this method is commonly referred to as the lowry method. Cuprous ions reduction of folin ciocalteu reagent produces a blue color that can be read at 650-750nm. The a mount of color produced is proportional to the amount of peptide bonds such as size, amount of protein/peptide

9. This method requires relatively large quantities of protein (1 - 20 mg protein / mL) for detection. Additionally, it is sensitive to a variety of nitrogen-containing substances that could be in the protein solution, thereby increasing the likelihood of erroneous results.

10. A standard curve is a type of graph used as a quantitative research technique. Standard curve for protein concentration is often created using known concentrations of bovine serum. The protein we will analyze is bovine serum albumin (BSA). Albumin is a serum protein that transports fatty acids and is important in maintaining plasma pH. In protein quantization assays, BSA serves as a reference protein that is used to construct protein standard curves. Other proteins can be used depending on the physical/chemical properties of your protein of interest.

11. The preparation of a standard curve is necessary to check whether the method of assaying a particular substances increases in a linear way with its concentration. The general formula for obtaining different concentrations of a solution by dilution with diluent is: C1V1=C2V2

12. Conc. mg/mlUnknown (ml)Protein standard (ml) D.W (ml)Tubes 000.51 0.40.10.42 0.80.20.33 1.20.30.24 1.60.40.15 20.506 00unknown After this, add 1 ml of biuret reagent to each tube and mix. Incubate the tubes for 30 mints at room temperture. Read at 540 nm on spectrophotometer. Make the standard curve and measure the concentration of unknown. After this, add 1 ml of biuret reagent to each tube and mix. Incubate the tubes for 30 mints at room temperture. Read at 540 nm on spectrophotometer. Make the standard curve and measure the concentration of unknown. Blank solution: A blank solution is a solution containing little to no analyte of interest, usually used to calibrate instruments such as a colorimeter.solutionanalytecolorimeter

13. According to beer_law The Beer-Lambert law (Beer’s law) mathematically establishes the relationship between concentration and absorbance in many photometric determinations. Beer’s law is expressed as A = abc The concentration of substance is directly proportional to the amount of light absorbed or inversely proportional to logarithm of the transmitted light. A: absorptivity constant for the substance B: length of the light path through the substance. Reference range for total proteins is : 66.6 to 81.4 g/l

14. Multiple samples with known properties are measured and graphed, which then allows the same properties to be determined for unknown samples by interpolation on the graph. The samples with known properties are the standards, and the graph is the standard curve.

15. Draw the points with protein concentrations as x values and the average absorbance as y values on a grid or graph paper Draw a straight line through the points Lookup the unknown protein concentration from the plot using the absorbance value of the unknown protein. signal concentration

17. The biuret protein assay is very stable and follows Beer’s law. Rather than make up a completely new standard graph, one standard (6 g/dL) was assayed. The absorbance of the standard was 0.400, and the absorbance of the unknown was 0.350. Determine the value of the unknown in g/dL? C 5.25 g/DL This method of calculation is acceptable as long as everything in the system, including the instrument and lot of reagents, remains the same. If anything in the system changes, a new standard graph should be done. Verification of linearity and/or calibration is required whenever a system changes or becomes unstable. Regulatory agencies often prescribe the condition of verification as well as the how often the linearity needs to be checked.

18. Sensitivity: lowest amount of analyte in a sample which can be detected. Specificity: is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. The linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample.”