Isoelectric Focusing Fundamental of Bioprocess Engineering Laboratory

1. Isoelectric FocusingFundamental of Bioprocess Engineering Laboratory Sally Lai Eric Guo Jerry Yin

2. Aim of experiment • To fractionise proteins using an electrophoretic technique according to their isoelectric point (pI) along a continuous pH gradient. • The proteins that are to be used: • Ovalbumin • Casein • Gluten

3. Theoretical Background • What are Proteins? • Net charges • Isoelectric point • Isoelectric Focusing

4. Proteins • A chain of amino acids • Amphoteric molecules; they carry either positive, negative or zero net charges • pH dependant for the net charge

5. Net Charge • Net charge of protein is the sum of all the negative and positive charges of its amino acid side chains and amino- and carboxyl- termini

6. Isoelectric Point (pI) • The point where the protein has a net charge of zero (that is, the sum of the negative and positive charges equal to zero) • Reached only when the pH = pI, as shown in the previous diagrams.

7. Isoelectric Focusing (IEF) • Is a protein purification and fractionation technique • Concentrates proteins at their pIs and allows proteins to be separated on the basis of very small charge differences • The separation occurs only under the influence of an electric field

8. Process of running IEF • Prepare the solutions • Prepare the sample and the protein standard into applicator • PhastSystem operations • Collect results

9. Prepare the solutions • Urea solution • The washing solution (30% methanol, 10% (v/v) acetic acid) • The stock solution (0.2% (w/v), 200mL) • The fixing solution (20% (v/v) 25mL trichloroacetic acid) toxic!! • The final stage solution (0.1% (w/v) CuSO4 over mixture of washing solution and stock solution) • Final IEF solution (to be used fresh)

10. Prepare the samples • Dissolve proteins into prepared Urea solutions • Fill the samples and standards to the 8 depressions on the Parafilm (2 for each) • Load sample applicator

11. PhastSystem operations • Place two gels onto the separation bed, remove plastic film • Insert the applicator • Run the sample with the PhastSystem programmed operations • Remove the gel into fresh final solution for staining.

12. Results • Experimental Failure: no results appeared for the 3 runs. • Sample results are used instead for calculations

13. Sample results

14. Useful data

15. Solving the problem Run 1a: y = 0.855x - 2.0364 Run 1b: y = 0.8635x - 2.0408

16. Discussion • Theoretical PI point: • Ovalbumin = 5.19 • Casein: 4.98 • Gluten = 7.64 • Blank result in our film • Possible problems

17. Solutions • Protein solutions • Protein solubility • Protein purity • Solvent • Stain solutions • Fix solution

18. Equipment • Equipment set up • Insufficient electrodes cleaning • Poor contact between electrode and gel • Dropping of Applicator arm • Damage of contact block and pin • PhaseSystem operate and programming problem • Bent applicator • Insufficient power/time

19. Cooling problem • PI point of protein is temperature dependant. • The performance of coolant will affect the mark positions. (May result the marks will be out of gel’s visible range)

20. Conclusion • Unexpected results occurred • Possible reasons • Solutions prepare • Equipments • Procedure

21. Recommendations • Use better buffer such as acid or detergent buffer to improve the proteins solubility • Prevent possible protein denature. • more accurate concentration. • Make the final solution and fix solution fresh

22. Recommendations (con.) • Use the equipment (PhastSystem) after it has been tested and prove the machine work as expect. • Program different and appropriate methods of IEF PhastSystem. • Make better experiment plan and implement accordingly. • Use more time to study the theory of the experiment before rush into lab work