4/10 application of protein isolation techniques

• Blood tests provide a snapshot of overall health and are often the first step in disease diagnosis.
• • Glucose: high in diabetes

  • Urea: high in kidney disease

  • LDL cholesterol: high in cardiovascular disease
• • Blood tests may be performed as part of a routine physical examination or because of the presence of specific symptoms
  • Blood tests will measure the levels of various components: blood cells and platelets, electrolytes, proteins, hormones, and certain minerals
• • Albumin: marker of liver and kidney function

  • Alkaline phosphatase (ALP): high levels can indicate liver or bone disorders

  • Alanine aminotransferase (ALT): high levels indicate liver damage

  • Aspartate amino transferase (AST): high levels indicate liver damage
• Aspartate aminotransferase (AST) = catalyzes the transfer of the amino group of aspartate to α-ketoglutarate

  Alanine aminotransferase (ALT) = catalyzes the transfer of the amino group of alanine to α-ketoglutarate
• Quantification of aspartate aminotransferase (AST) is coupled to NADH levels

  • Malate dehydrogenase (MD) catalyzes this reaction to convert oxaloacetate to malate and in the process converts NADH to NAD+

  • NADH in solution produces a significant absorbance peak at 340 nm, while NAD+ has virtually no absorbance at this wavelength

  • Measuring absorbance at 340 nm indicates NADH levels, which is a proxy for AST activity
• Quantification of alanine aminotransferase (ALT) is coupled to NADH levels

  • Lactate dehydrogenase (LDH) catalyzes this reaction to convert pyruvate to lactate and in the process converts NADH to NAD+

  • NADH in solution produces a significant absorbance peak at 340 nm, while NAD+ has virtually no absorbance at this wavelength

  • Measuring absorbance at 340 nm indicates NADH levels, which is a proxy for ALT activity
• Protein purification is often an essential first step in their quantification and understanding their function

  • Proteins can be purified

  – performed by subjecting an impure mixture of starting material to a series of separations based on physical properties such as size and charge

  – requires a test, or assay, that determines whether the protein of interest is present
• Analyzing a purification scheme

  • To analyze how a purification scheme is working, the amount of total protein present in the mixture being assayed must be known.

  • specific activity = the ratio of enzyme activity to the amount of protein in the mixture.

  • The overall goal of the purification is to maximize the specific activity.

  • increase purity, enzymatic activity, efficiency, specific activity
• Proteins must be released from the cell to be purified

  STEPS

  • 1: disrupt the cell membranes of intact cells to form a homogenate ...lyse membrane, release cellular components

  ex. mitochondria , nucleus, and separate these via centrifuge

  
  

  • 2: centrifuge the homogenate at low speed to yield a pellet w/ heavy material and lighter supernatant

  
  

  • 3: centrifuge supernatant at higher centrifugal force to yield another pellet and supernatant

  – differential centrifugation is repeated many times to yield several fractions of decreasing density.
• In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it.
• LOW SPEED CENTRIFUGATION

  • PELLET CONTAINS ...
  • whole cell
  • nuclei
  • cytoskeletons
• MEDIUM SPEED CENTRIFUGATION

  • PELLET CONTAINS ...
  • mitochondria
  • lysosomes
  • peroxisomes
• HIGH SPEED CENTRIFUGATION

  • PELLET CONTAINS ...
  • microsomes
  • small vesicles
• VERY HIGH SPEED

  pellet contains...

  • ribosomes
  • viruses
  • large macromolecules
• velocity sedimentation

  • subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose.
  • After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom
• equilibrium sedimentation

  • subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches that of their surroundings.
  • much steeper sucrose gradient
• salting out = effect by which most proteins are less soluble at high salt concentrations

  • The salt concentration at which a protein precipitates differs from one protein to another.

  • method for precipitating proteins for collection
• dialysis separates proteins from smaller molecules

  • through semipermeable membrane (cellulose membrane with pores)
  • molecules larger than pore diameter remain inside dialysis bag
  • smaller molecules and ions diffuse down concentration gradients and go into solution outside the bag
  • picture a filter
  • separation....but not specific way to isolate proteins
  • by sizing
• separation of molecules by Column chromatography

  • main way of separation
  • The sample, (a solution containing a mixture of different molecules), goes on top of a cylindrical glass or plastic column filled with a permeable gel matrix, like cellulose.
  • A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. Bc various components of the sample travel at different rates through the column, they r fractionated into different tubes.
  • the higher u are, the higher affinity to column --> slow to diffuse out ....IMAGINE: TRAFFIC
• gel filtration chromatography

  • separates proteins by size
  • column is filled with porous beads
  • sample is applied to top of column
  • slow rate as passes through column pores/beads
  • SMALL = SLOW
  • LARGE = FAST
  • carbohydrate polymer bead
  • small molecules enter the aq spaces within beads ...EXIT LAST
  • large molecules cannot enter beads...EXIT COLUMN FIRST
• protein purification by gel filtration chromatography

  • homogenate of cells is fractionated by passing impure protein through the matrix of the gel-filtration column.
  • small beads that form the matrix are inert but porous
  • molecules that are small enough to penetrate into matrix beads are delayed and travel slowly through the column than larger molecules that cannot penetrate
  • Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various masses.
• ION EXCHANGE CHROMATOGRAPHY

  • SEPARATION PROTEIN BY CHARGE
  • column filled with charged beads, sample is applied at top of column
  • CATION EXCHANGE = negatively charged beads
  • ANION EXCHANGE= POSITIVELY CHARGED beads
  • same charge - will exit column quickly ..
  • different charge - opposites attract so will be stuck. ultimately slowly released by increased salt concentration of buffer that is passed through the column
• Column washing


  Remove all unbound contaminants
• affinity chromatography by LIGAND AFFINITY

  • takes advantage of the fact that some proteins have a high affinity for specific molecules called ligands
  • column is filled with beads attached to specific ligand
  • no affinity = pass quick
  • increased affinity = slow
• LIGAND AFFINITY

  • affinity chromatography
  • some proteins have a high affinity for specific molecules called ligands
  • column is filled with beads attached to the specific ligand
  • IF NO AFFINITY = PASS QUICK
  • IF HIGH AFFINITY, SLOW
  • HOW IS IT ULTIMATELY RELEASE ?
  • by passing a solution enriched in the ligand to which the protein is bound through the column ex. free glucose

  
  

  • chromatography graph = two primary peaks
  • one large one for main who escaped
  • one small for leftovers that got stuck
• high performance liquid chromatography

  • uses fine beads in columns and pressure to move the liquid through column
  • leads to sharper separations between proteins + more rapid separation
• HPLC

  • tightly packed column , uses high pressure and flow rate .. quick
  • better, quicker separation for protein
  • lots of samples
• gel electrophoresis

  • separates proteins by size
  • with a net charge by applying electric field

  smaller --> travel quickly

  medium at medium

  larger --> slower speed
• chemical composition of gel electrophoresis

  • polyacrylamide gels
  • highly cross linked 3d mesh
• SDS-PAGE

  • standard approach for protein separation
  • allows accurate determination of mass
  • SDS= anionic detergent that denatures proteins
  • 1 molecule of SDS binds for every 2 amino acids
  • uses DYE CALLED COOMASSIE BLUE to stain them after electrophoresis
  • binds to basic and hydrophobic amino acid residues
  • electrophoresis can determine protein mass
  • ex. if ubiquitin was added to a protein, it would be higher up on the column bc it adds mass and it would travel slower than other ones.
• staining of proteins after electrophoresis

  • coomassie blue dye stain
  • binds to basic and hydrophobic amino acid residues
• electrophoresis can determine protein mass , faster = overall size small
• polyclonal antibodies - heterogenous mixtures of antibodies

  • derived from multiple antibody producing cell populations
  • each antibody is specific for one of the various epitopes on an antigen
• monoclonal antibodies = identical antibodies produced by clones of single antibody producing cell
• immortal cell lines produce Monoclonal antibodies
• western blotting - permits the detection and quantification of proteins separated by gel electrophoresis . separated in an SDS page gel - transferred to a polymer , stained with primary antibody, stained with secondary antibody
• coomassie blue, u see all the lines. but western blotting u see only one: only protein of interest where the antibody binded
• primary antibody: an antibody specific for the protein

  secondary antibody: antibody specific for primary antibody shapes

  • attached to an enzyme that gametes chemiliuminescent product or contains fluorescent tag to enable identification and quantification
• synovial sarcoma - rare pediatric cancer driven by a single genetic event: chromosomal translocation that produces ss18-ssx oncogenic fusion protein.