Isoelectric Focusing Fundamental of Bioprocess Engineering Laboratory

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Isoelectric FocusingFundamental of Bioprocess
Engineering Laboratory

• Sally Lai
• Eric Guo
• Jerry Yin

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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

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Proteins

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

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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

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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.

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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

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Process of running IEF

• Prepare the solutions
• Prepare the sample and the protein standard into
  applicator
• PhastSystem operations
• Collect results

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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)

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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

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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.

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Results

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

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Sample results
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Useful data
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Solving the problem
Run 1a y 0.855x - 2.0364
Run 1b y 0.8635x - 2.0408
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Discussion

• Theoretical PI point
• Ovalbumin 5.19
• Casein 4.98
• Gluten 7.64
• Blank result in our film
• Possible problems

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Solutions

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

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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

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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
  gels visible range)

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Conclusion

• Unexpected results occurred
• Possible reasons
• Solutions prepare
• Equipments
• Procedure

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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

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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