Isoelectric Focusing Fundamental of Bioprocess Engineering Laboratory Sally Lai Eric Guo Jerry Yin

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

Theoretical Background What are Proteins? Net charges Isoelectric point Isoelectric Focusing

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

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

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.

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

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

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)

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

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.

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

Sample results

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

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 = 0.8635x - 2.0408 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

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

Solutions Protein solutions Stain solutions Fix solution Protein solubility Protein purity Solvent Stain solutions Fix solution

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

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)

Conclusion Unexpected results occurred Possible reasons Solutions prepare Equipments Procedure

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

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