Protein Assay by Turbidimetric Method The turbidity produced when proteins are mixed with low concentrations of a protein precipitant this method from a standard curve; (a graph where the quantity of solute is known). can be used as an index of protein concentration. Protein concentration may be determined by Instruments and supplies Pipette (100 L and I mL) Pipette tips Parafilm Cuvettes Test tubes Spectrophotometer 1 mg/ml Bovine serum albumin (BSA) 20%. Trichloroacetic acid (TCA) Distilled water Samples of unknown concentration: liver extract, affinity chromatography fraction, nuclear and mitochondrial proteins Experimental procedure 1. Place I ml distilled water in nine test tubes labelled through 9. 2. Remove the following volumes of water from the test tubes 1 through 9 respectively and replace with an equal volume of BSA solution of concentration 1 mg/ml. Each tube should have a total of 1 ml of solution (see Table below) Tube # Water (l.) 1 mg/mL BSA (L.) 1000 L OL 2 990 L 10 L 975 uL 25 ul 4 950 L 5 925 ul 75 l 6 900 L 100 L 7 850 L 150 L 8 800 uL 200 ul 9 750 ul. 250 L The BSA is used here as a representative protein. 25 3. For each unknown sample concentration to be assayed, place 1 mL of distilled water in a labelled test tube. Remove 50 L of water and replace it with an equal volume of sample. *This volume may be increased or decreased so that the absorbance value lies between those for the 25 uL anc 250 uL standards. In any case, the volume of the sample and the distilled water should be 1 ml Once you have found a useful dilution for each of your important samples, repeat the dilutic and assay the sample again. This is called “performing a replicate”; the replicates will allow yo to determine the protein concentration in your samples with more confidence. قيا 50 L 39 10 Affinity chromatography fraction Nuclear proteins 4. Add 2 ml of 20% TCA to each tube (both standard and sample). Mix and allow the tubes to stand at room temperature for 10 minutes. 5. Mix the 'blank' (Tube 1) in a vortex mixer, transfer to a cuvette and use it to adjust the spectrophotometer to display zero absorbance at 600 nm. 6. Pour the contents of the cuvette into a 'waste' beaker and leave the cuvette inverted on “Kimwipe' to drain. 7. Repeat steps 5 and 6 for Tubes 2 to 11 (except for adjusting the spectrophotometer; note the absorbance). Once you have measured the absorbance values for the standard curve and for your unknown protein samples, you will need to use the standard curve to determine the protein concentrations for your unknowns. 8. Construct a standard curve. a. Using the concentration and the volume of BSA solution, calculate the quantity of BSA (Hg) contained in each standard tube. b. Plot a graph absorbance (on the ordinate) vs the quantity of protein (on the abscissa). c. Draw the best fit straight line. It should pass through the origin. Note: The best method is to plot the values obtained for the standard curve, determine the slope of the best-fit line, and then use the equation of the line to give the protein concentrations for your unknown samples. 9. Determine the protein concentration of the sample. a. Using the standard curve, determine the quantity of protein equivalent to the absorbance of the sample b. Using the protein quantity determined above and the volume of undiluted samples (HL) used in the assay, calculate the protein concentration of the samples. Protein concentration is often expressed in ug or mg/mL. In other words, it is the quantity of protein in 1 mL of the undiluted sample. 40 5 Data Sheet b.pdf 1 a Sheet b.pdf 5 Data Sheet b.pdf (1.36 MB) Tube # Volume Amount of used (ul) BSA (ug) OUL 2 10 L 25 uL 4 50 L 5 75 uL 100 L 7 150 L 8 200 L 9 250 L اقوا Absorbance at 600 nm 0 0.056 0.165 0.287 0.401 0.579 0.801 1.098 1.364 6 Sample Tube # Volume Absorbance Quantity of protein (ng, Protein used (L) at 600 nm extrapolated from concentration standard curve) (mg/mL) 25 0.332 25 0.923 10 11 JS o E