isoelectric focusing fundamental of bioprocess engineering laboratory sally lai eric guo jerry yin
TRANSCRIPT
Isoelectric FocusingFundamental 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 IEF1. Prepare the solutions
2. Prepare the sample and the protein standard into applicator
3. PhastSystem operations
4. 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 stain
ing.
Results
Experimental Failure: no results appeared for the 3 runs.
Sample results are used instead for calculations
Sample results
Useful data
Protein standard Theoretical pI Distance from the base line
Run 1 Run 2
Amyloglucosidase 3.6 1.1 1.1
Glucose oxidase 4.2 1.4 1.4
Trypsin Inhibitor 4.6 1.85 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 3.9
Myoglobin (basic) 7.2 4 4.1
Lentil Lectin (acidic) 8.2 5.2 5.25
Lentil Lectin (middle) 8.6 5.25 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 6.95
Solving the problemdistance pI
y x
3.05 5.95 b
2.50 5.31 c
4.70 7.88 d
3.90 6.94 e
Theoretical pI vs. Distance
y = 0.855x - 2.0364
R2 = 0.9945
0
1
2
3
4
5
6
7
8
0 5 10 15
Theoretical pI
Dis
tan
ce
(c
m)
Run 1
Linear (Run 1)
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
Run 1a: y = 0.855x - 2.0364
Run 1b: y = 0.8635x - 2.0408Theoretical pI vs Distance
y = 0.8635x - 2.0408
R2 = 0.9952
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12
Theoretical pI
Dis
tan
ce (
cm)
Run 2
Linear (Run 2)
Discussion
Theoretical PI point:– Ovalbumin = 5.19– Casein: 4.98– Gluten = 7.64
Blank result in our film Possible problems
Solutions
Protein solutions– 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 fres
h
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