Protein Techniques

• Central Dogma

  
• Proteins - Large molecules composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein.
• Functions of Proteins

  •Growth and maintenance

  •Energy source 4 calories/g

  •Catalysts in biochemical reactions – enzymes

  •Transport – neurotransmitters, hemoglobin

  •Messenger molecules – hormones

  •Provide structure – keratin, elastin, collagen

  •Defense – antibodies

  •Homeostasis - a relatively stable equilibrium

  physiological state
• Anionic

  • charge: negative
  • shape: globular
  • size: small, medium, large
  • role: Metabolic, structural, regulatory
• Cationic

  • charge: positive
  • shape: fibrous
  • size: small, medium, large
  • role: Metabolic, structural, regulatory
• Protein Sources

  • Meat
  • Milk and milk products
  • Eggs
  • Legumes
  • Nuts
• Extraction of Soluble Proteins

  • Materials: sample protein source, buffer - chemical reagents, dH20
  • Equipment: analytical balance, pH meter, hot plate stirrer, centrifuge
  • Other laboratory wares: glass wares - beakers, grad cylinder, scissors, weighing boats
• Buffers - a solution containing appreciable amounts of a

  weak conjugate acid-base pair is called a buffer

  solution, or a buffer.
• Buffer solutions - resist a change in pH when small

  amounts of a strong acid or a strong base are

  added
• Phosphate Buffer - consists of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate
• Calculations:

  • Weight = Molecular weight x Molarity x Volume

  • C1V1 = C2V2
• pH meter - uses an electrode to measure the pH of a solution.
• Calibrating and using the pH meter

  1. Plug pH meter at least 15 minutes before using
  2. Rinse the electrode with distilled water
  3. Blot in dry and immerse in standard buffer 1
  4. Read pH of standard buffer 1
  5. Rinse the electrode with dH2O, blot dry and immerse in second standard buffer.
  6. Read pH of standard buffer 2.
  7. Rinse the electrode with dH2O, blot dry
  8. Immerse electrode in solution to be measured
  9. Adjust pH of the solution using acid or basic solutions.
  10. Clean the electrode after use and keep in storage solution.
• Analytical Balance - accurate and precise instruments that measure masses and require a draft-free location on a solid bench that is free ofvibrations.
• Tips on Proper Care

  • Do not bump or place objects on the bench after zeroing the balance.
  • Mass powders on paper or dishes. Handle objects with tongs, tweezers, gloves, or paper to prevent fingerprints.
  • Let hot objects cool before massing.
  • Mass hygroscopic materials rapidly since they will absorb water during massing.
  • When making repetitive massings always use the same procedure.
  • DO NOT go off and leave spilled chemicals on or around the balance! clean it up in a proper manner, re-calibrate if necessary.
• Sample preparation (Plant)

  • Plant parts – leaf, stem, roots, flowers
  • wash with tap water to remove dirt
  • rinse with distilled water
  • blot dry with paper towel
  • grind under liquid nitrogen
  • use immediately or store at -20°C or -80°C
• Sample preparation (Plant)Seeds

  • dehull
  • grind to a fine powder
  • use immediately of store at 4°C
• Protein Extraction – Soluble Proteins

  1. Homogenize sample in extraction buffer
  2. Filter through 4 layers of cheese cloth or miracloth
  3. Transfer in centrifuge tubes or bottles as needed
  4. Centrifuge
  5. Collect supernatant
  6. Keep on ice until use or store in ref/freezer
• Protein Extraction Steps Summary

  1. Preparation of Buffers for Protein Extraction– Phosphate Buffer
  2. Sample preparation - Plant
  3. Protein Extraction – Soluble Proteins
  4. Centrifugation
• Centrifugation - a technique of separating

  substances which involves the application of

  centrifugal force.

  • Pulling away from the center

  • The particles are separated from a solution

  according to their size, shape, density, the viscosity

  of the medium and rotor speed.