1. Quantitative tests for proteins

2. Introduction
Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure.
Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Daltons.

3. Aim and significance Aim: Significance:
To perform biuret test for proteins.
To estimate the amount of total proteins in a sample.
To make standard curve.
Significance:
The quantization of protein content is important and has many applications in clinical laboratory practices and in research especially in the field of biochemistry. The accurate quantization of protein content is a critical step in protein analysis.

4. Biuret test
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. Biuret reagent
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.
Cupric sulfate pentahydrate (1.5g) and sodium potassium tartrate (6 g) were weighed and dissolved in 500 mL distilled water.
To the resulting solution were added 300 mL of 10 % NaOH with constant stirring.
The resulting mixture was then transferred quantitatively into a 1 L volumetric flask and diluted to mark with distilled water.

6. Principle
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 nm.
The a mount of color produced is proportional to the amount of peptide bonds such as size, amount of protein/peptide

9. Biuret test
This method requires relatively large quantities of protein ( 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. Standard curve
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.
The relationship of absorbance to protein concentration is approximately linear within the indicated range of protein concentrations for the reference solutions.
Plot the absorbencies of the reference solutions against protein concentrations and use linear regression to establish the standard curve. Calculate the correlation coefficient for the standard curve. A suitable system is one that yields a line having a correlation coefficient not less than From the standard curve and the absorbance of the test solution, determine the concentration of protein in the test solution.

11. Standard curve
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. Procedure Conc. mg/ml Unknown (ml) Protein standard (ml) D.W (ml)
Tubes
0.5 1
0.4
0.1 2
0.8
0.2
0.3 3
1.2 4
1.6 5 6
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.

13. How to calculate the concentration
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. How to make a standard curve
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 a straight line through the points
Cont….
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. 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.”