1. EPPING, LQB381 Flow Diagrams Dr Ron Epping LQB381 Unit Coordinator 1991 - 2012

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3. EPPING, LQB381  Simple diagrams help you organise your mind and understand how dilution factors apply to an experiment. Try working backwards using AMOUNTS (g or moles) from the results of the test sample to calculate the AMOUNT in the original sample. Here is an example 100 uL of a protein extract is diluted 1/20 with saline and then assayed for protein as follows: 100 uL of a protein extract is diluted 1/20 with saline and then assayed for protein as follows: 250 μL of the test sample (diluted extract) is mixed with 100 μL of Reagent A and 500 μL of Reagent B. The solution is then diluted to 3 mL with d.water and the absorbance measured. From comparison with plotted standards on a standard curve. the 3 mL sample is found to contain 1.5 mg of protein. 250 μL of the test sample (diluted extract) is mixed with 100 μL of Reagent A and 500 μL of Reagent B. The solution is then diluted to 3 mL with d.water and the absorbance measured. From comparison with plotted standards on a standard curve. the 3 mL sample is found to contain 1.5 mg of protein. What is the protein concentration of the original solution in mg/mL? What is the protein concentration of the original solution in mg/mL?

4. EPPING, LQB381 Flow Diagram: Example Protein Solution PROTEIN SOLUTION 1.0 mL

5. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL PROTEIN SOLUTION 0.1 mL Original Sample 1.0 mL

6. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 1.0 mL

7. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL0.25 mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 1.0 mL2.0 mL

8. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL0.25 mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 0.25 mL Test Sample 1.0 mL2.0 mL

9. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL0.25 mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 0.25 mL Test Sample 1.0 mL2.0 mL0.25 mL

10. EPPING, LQB381 Flow Diagram: Example Protein Solution 0.1mL0.25 mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 0.25 mL Test Sample 1.0 mL2.0 mL0.25 mL ASSAY PROCEDURE + 0.1 mL A + 0.5 mL B + 0.15 mL H 2 O 1.0 mL ABSORBANCE 0.25 mL 1.5 mg Read from the standard curve

11. EPPING, LQB381 Flow Diagram: Example 0.1mL0.25 mL PROTEIN SOLUTION 0.1 mL Original Sample 1.9 mL saline 0.25 mL Test Sample 1.0 mL2.0 mL 0.25 mL CALCULATE USING AMOUNTS: 1.5 x 10 -3 g x 2.0 / 0.25 x 1.0 / 0.1 = 0.12 g in 1 mL original sample = 120 mg/mL ASSAY PROCEDURE + 0.1 mL A + 0.5 mL B + 0.15 mL H 2 O 1.0 mL ABSORBANCE 1.5 mg

12. EPPING, LQB381