1. Isoelectric Focusing Fundamental 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. Distance from the base line
Useful data
Protein standard
Theoretical pI
Distance from the base line
Run 1
Run 2
Amyloglucosidase
3.6
1.1
Glucose oxidase
4.2
1.4
Trypsin Inhibitor
4.6
1.85
β-lactoglobulin
5.1
2.25
2.3
Carbonic anhydrase III
5.4
2.6
2.65
Carbonic anhydrase II
5.9
3.25
3.3
Carbonic anhydrase I
6.6
3.55
3.7
Myoglobin (acidic)
6.8
3.9
Myoglobin (basic)
7.2 4
4.1
Lentil Lectin (acidic)
8.2
5.2
5.25
Lentil Lectin (middle)
8.6
5.35
Lentil Lectin (basic)
8.8
5.3
5.45
Ribonuclease A
9.5
6.2
6.25
Cytochrome C
10.6
6.95

15. Solving the problem Run 1a: y = 0.855x - 2.0364
distance pI y x
3.05
5.95 b
2.50
5.31 c
4.70
7.88 d
3.90
6.94 e
Run 1b: y = x
6.70
10.12 g
5.10
8.27 h
4.80
7.92 I
5.65
8.91 J
1.65
4.27 K

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

17. Solutions Protein solutions Stain solutions Fix solution
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