CHM4116L - Protein analysis

• Several conditions must be controlled to keep proteins stable

  1. pH - optimum or denatured
  2. Temperature - optimum or denatured
  3. Presence of degradative enzymes - proteases leading to be hydrolyzed
  4. Adsorption to surfaces - tendency to absorb to surfaces
  5. Long term storage - conditions and protocols used
• Protein Isolation and Purification steps from a biological sample

  1. Cell disruption - destroy cell to release components of the cell
  2. Fractionalization - such as centrifugation which separates components based on its physical feature (size, etc)
  3. Crude sample preparation - how to prepare the sample that contains the biological activity
  4. Purification - an optional stage
• Cell disruption can be achieved in any one of the following methods

  • Detergents
  • destabilize hydrophobic interactions of lipid membranes
  • Lysozyme
  • Proteases - hydrolyze membrane proteins for efficient cell destruction
  • Ultrasonication
  • High frequency waves and vibration rates, destabilizing the interactions between cells
  • Osmotic shock
  • A hypotonic solution disrupts and lyse the cell when shocked
  • Mechanical stress (Homogenization)
  • Pressure + sharped objects + stress
• Cell disruption techniques are used to isolate a biological component (crude extract) in a cell
• Preparation of crude extract

  • Lyse cells in lysis buffer containing inhibitors of both proteases and phosphatases
  • Phosphatases - inhibited to observe if a phosphorylated/dephosphorylated sample is seen in a protein
  • Centrifuge lysate to remove membranous cellular debris
  • Remove insoluble content to proceed on homogenous media analysis
  • Determine protein concentration in ug/ul of the lysate
• Components of the Lysis Buffer

  • 50mM Tris HCl ; pH of 8.0 - pH friendly to most protein
  • 150mM NaCl, isotonicity, having equal tension or tonicity
  • Detergents - disrupting lipid bilayers, capable of solubilizing H. proteins
  • Dithiothreitol - reducing agent
  • Prevents inappropriate oxidation of reduced cysteines to disulfide bonds
  • Prevents covalent aggregation and precipitation of proteins that are not covalently linked in vivo
• Detergents

  • NP-40 (non-ionic, good solubilization, weakly denaturing)
  • Deoxycholate (a bile acid, ionic, moderately denaturing)
  • SDS (synthetic, ionic, excellent solubilization, strongly denaturing)
• Components of the lysis buffer

  • Protease inhibitors
  • Prevent protein degradation and thereby allow more accurate determination of molecular weight
  • Phosphatase inhibitors
  • Inhibitors prevent enzymatic removal of phosphates from phosphorylated proteins during extract preparation
• Protease inhibitors

  • PMSF (phenylmethanesulfonylfluoride)- inhibits serine proteases
  • Leupeptins
  • Tripeptides produced by various species of actinomycetes
  • L-leucyl-L-leucyl-D-argininal
  • modified at NH-terminus by acetyl or propionyl
  • Found in pancreas and lung, among other tissues
  • Natural inhibitor of various extra and intracellular proteases
• Phosphatase inhibitors

  • Inhibitors prevent enzymatic removal of phosphates from phosphorylated proteins using extract preparation
  • Phosphorylated and dephosphorylated proteins migrate differently during SDS-PAGE
  • Useful information can be gained by knowing whether or not a protein is phosphorylated in vivo in given cells under specific conditions
• Phosphatase inhibitors

  • NaF
  • Na3VO4
• Boiling samples can denature proteins even without a protease inhibitor; filling with a protease inhibitor selectively denatures a protein
• Protein Purification is a stepwise process

  A) Solubility

  B) Ionic Charge

  C) Polarity

  D) Size

  E) Binding Specificity

  
• Salting out separates proteins by their solubility

  • The protein solvent interaction changes by adding a salt concentration
  • The salt would interact with the solvent
  • Protein interacts with another protein, salting out occurs particle size increase and it forms aggregates
• Salting in

  • low and high salt concentration becomes soluble
  • middle concentration becomes insoluble
  • Water at high amount cannot interact with the salt thus it can interact again with protein
• Salting in/Salting out

  • Changing salt concentration on protein protein interaction will be changed
  • A dominated protein protein interaction can precipitate out of the solution
• Different proteins are least soluble at their varying isoelectric points
• Chromatography involves interaction with mobile and stationary phase

  • Ion exchange chromatography separates anions and cations
• Ion exchange chromatography

  • Charges of protein
  • Mixture of samples w/ ion exchanger (+ or -) and it retains opposite charge in the S.P
  • Changing the salt conc. can change elution time because sample would be stuck to the S.P
• Ion exchanger utilizes

  • Anion exchanger is a matrix attached to diethylaminoethyl (DEAE) groups
  • Matrix-CH2-CH2-NH(CH2CH3)^2+
  • Cation exchanger is a matrix attached to carboxymethyl (CM) groups
  • Matrix-CH2COO-
• Hydrophobic Interaction chromatography (Normal phased chromatography, purifies nonpolar molecules

  • The matrix material is lightly substituted with octyl or phenyl group
• Gel filtration chromatography separates molecules according to size
• Gel filtration chromatography separates molecules according to size

  • S.P - Beads in which pores are created
  • at large molecules, it has less interactions with the beads, which is eluted first, and vice versa
  • small molecules undergo many pores in the gel beads
• Affinity Chromatography exploits specific binding behavior between protein and a compound

  • Immuno affinity chromatography - antibody is attached to the matrix (SP) in order to purify the protein (antigen) against which the antibody is raised. Wash column with another antigen to displace the protein in the antibody.
  • Metal chelate affinity chromatography - a divalent ion such as Zn2+ or Ni2+ is attached to the matrix so that metal chelating bearing groups can be specifically retained. Supply with another ligand to remove the proteins in the chelate
• Proteins are quantified by assays

  • specific protocols are utilized
• ELISA

  • Enzyme-Linked immunosorbent assay
  • Gold standard
  • One technique used to detect the presence of a protein in a mixture by binding to its corresponding antibodies
• Quantitative estimation of proteins

  • Protein concentrations can be estimated by spectroscopy at 280 nm
  • Biuret Method
  • Folin-Lowry method of protein assay
  • BCA (Bicinchoninic acid) protein assay
  • Bradford assay
• 280 nm

  • maximum absorbance of aromatic amino acids (Phe, Trp, Tyr)
  • not the gold standard but can estimate the number of proteins
• Biuret Method

  • Dilute CuSO4 + NaOH at alkaline pH, reacts with proteins - tripeptide
  • forms violet complex read at 540 nm
  • minimum of two peptides
  • limit of detection 1 - 20 mg protein/mL
• Folin-Lowry method of protein assay

  • Folin-Ciocalteu reagent
  • phosphomolybdic acid and tungstate
  • Aromatic amino acids, Tyr, Trp present in proteins react with these and produce a dark blue color
  • Color formed is due to the reaction of alkaline copper with protein as in the biuret test and the reduction of phosphomolybdate by tyrosine and tryptophan present in the protein
  • Concentration of the reduced folin reagent is measured by absorbance at 750 nm
  • LOD : 1-200 ug/mL
• BCA (Bicinchoninic Acid) Protein assay

  • proteins can reduce Cu2+ to Cu1+ in an alkaline solution (the biuret reaction) and result in a purple color formation by biocinchoninic acid at a maximum absorbance of 562 nm
  • The reduction of copper is mainly caused by four amino acid residues including Cysteine, cystine, tyrosine, and tyrptophan that are present in the protein molecules
  • LOD - 20 to 2000 ug/mL of protein
• Bradford assay -reddish brown to blue form

  • simple, fast, inexpensive, highly sensitive
  • Uses the acidified Coomassie brilliant blue G-250 dye reagent (binds electrostatically with arginine residues in anionic form by pi-stacking interactions with aromatic AA's)
  • Can also interact with positively charged amino acids but with the lowest interaction
  • When the dye binds to protein, it is converted blue form detected at 595 nm
  • Intensity of color (measured by absorbance) is directly proportional to the concentration of protein
  • LOD : 2 to 2000 ug/mL of protein
• Qualitative Techniques

  1. Electrophoresis
• Electrophoresis

  • a general term that describes the migration and separation of charged particles (ions) under the influence of an electric field
  • an electric current is used to move molecules to be separated through a gel
  • ions have different migration rates depending on their total charge, size, and shape
• Molecular sieve - separates molecules based on a specific size/charge ratio
• Electrophoresis

  • the technique is used particularly for macromolecules, such as proteins and nucleic acid
  • have ionizable group, which at given pH exist in a solution as electrically charged species such as cations and anions
  • under the influence of electric field, these charged particle will migrate either to anode or cathode depending on the nature of the net charge
• Gel electrophoresis

  • can be made from gels such as agarose or polyacrylamide
• Agarose

  • a complex sugar from red seaweed
  • it is commonly used in foods (ice cream, and jellies) and many biological mediums
  • it has a large pore size for separating large molecules quickly

  Polyacrylamide

  • chain of acrylamide molecules
  • it is often used to make plastics and rubber
  • it has a small pore size good for separating small molecules
• Gel electrophoresis

  • DNA/Protein is applied to a macromolecular cage or gel such as agarose or polyacrylamide, its migration under the pull of current is impeded
  • nucleic acids, large proteins, and protein complex is usually applied to agarose
  • most proteins and small oligonucleotides can be used in polyacrylamide
  • both are relatively electrically neutral
• The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration

  • The concentration of gel/buffer will affect the resolution of fragments of different size ranges
  • Increasing the concentration of the medium increases the separation/resolution and vice versa