WEEK 1

• Buffers


  Establishes the pH of the system and the electrical charge on the solute
• Ideal buffer properties

  • Preserves the analytes ability to dissolve
  • Keeps the buffering capacity constant throughout analysis
  • Shouldn't prevent the intended analytes from being detected
  • Achieves the appropriate range separation
• Starch Gel

  1. First gel medium used for electrophoresis

  2. Facilitates the separation of proteins based on charge-to-mass ratio and molecular size

  3. Colloidal suspension prepared by boiling the suspension of starch granules in a buffer

  4. Sets as a semisolid gel due to the intertwining of the branched chains of amylopectin

  5. Petroleum jelly added to avoid swelling and shrinking

  6. Sharp zones and high resolving power can be achieved
• Support Media

  • Includes starch, polyacrylamide, agarose, and cellulose acetate membranes in the form of sheets, slabs, and columns
  • Colloid that contains more than 90% water
  • Serves as a molecular sieve through which molecules are separated
  • Small molecules can pass through it because it is porous, while larger molecules cannot
  • Electrical neutrality is required
• Agarose


  1. Isolated from red seaweeds, contains agarose, a linear polymer composed of galactose and 3,6-anhydro-galactose chains

  2. Kept as a dry powder in storage

  3. Controls the pore size of the gel

  4. Used at 0.8% (W/V) to 5% (W/V) to distinguish between DNA and RNA molecules

  5. Relatively poor resolution compared to polyacrylamide gels

  6. Forms stable gels and considered perfect for gel electrophoresis
• Cellulose Acetate

  1. First developed for separating the protein hemoglobin found in red blood cells

  2. Filter papers made entirely of cellulose and acetylated to produce cellulose acetate

  3. Glucose ring's C-3 and C-6 locations typically where acetylation occurs

  4. Bigger pores compared to other common electrophoretic matrices like agarose and polyacrylamide
• Polyacrylamide


  1. Clear, transparent gel formed by the copolymerization of acrylamide monomers in the presence of the crosslinking agent N, N-methylene-bis-acrylamide

  2. Acrylamide concentration must be in proportion to its crosslinking agent, controls the size of the pores in polyacrylamide gels
• Types of Buffers

  • Acidic Buffers: Citrate, Acetate, Formate, Phosphate
  • Basic Buffers: Tric, Boarate, Tricine
• Separating DNA and proteins typically requires a small amount of acrylamide gel
• Separating DNA and proteins typically requires a small amount of acrylamide gel (3%-15%)
• Gel Caster and Comb

  • Gel is poured into a gel caster stored inside the apparatus
  • Wells are placed using a comb for sample loading
• In SODIUM DODECYL SULFATE (SDS)-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE), proteins are separated under denatured conditions according to their size, where a higher percentage of acrylamide gel (10%-20%) is typically used
• Electrodes

  • Two platinum electrodes help separate molecules by attracting charges with opposite charges
  • Anode binds positive ions, cathode binds negative ions
• Electrophoresis Chamber


  • Plastic container or tank filled with a buffer to prevent biomolecule movement
  • Transparent lid to see the migration process
  • Wired to a power supply
• Containers for Staining and Destaining Gel

  • Can be accomplished using trays and containers
  • Available lidded boxes and open-form boxes
  • Typically have a propylene base
  • Transparent and resistant to chemicals and stains
• Parts of Gel Electrophoresis

  1. ELECTROPHORESIS CHAMBER

  2. CONTAINERS FOR STAINING AND DESTAINING GEL

  3. ELECTRODES

  4. GEL CASTER AND COMB
• Types of Electrophoresis

  • Paper gel electrophoresis
  • Agarose gel electrophoresis
  • Polyacrylamide gel electrophoresis
  • Pulsed-field gel electrophoresis (PFGE)
  • Sds-page
  • 2d-electrophoresis
  • Immunoelectrophoresis (rocket electrophoresis)
  • Difference gel electrophoresis (DIGE)
• Two most common types of Gel Electrophoresis

  • Agarose gel electrophoresis
  • Polyacrylamide gel electrophoresis
• Polyacrylamide Gel Electrophoresis

  • Used at a concentration of up to 3-30% (pH range: 4-9.0)
  • Higher concentration for protein separation, lower for DNA separation
  • Reliable and accurate porosity
  • Applications include calculating DNA's molecular weight, DNA sequencing, studying DNA purity, analyzing recombinant DNA molecules, separating RNA molecules, and measuring RNA's molecular weight
• Paper Gel Electrophoresis is used to investigate serum and other bodily fluids in clinical settings
• Agarose Gel Electrophoresis


  • Concentration determines resolution
  • Suited for separating DNA fragments ranging from 100 base pairs to 20 kilobase pairs
  • Applies to the electrophoretic separation of proteins
  • Can be used for isoelectric focusing
• Pulsed-Field Gel Electrophoresis (PFGE) was introduced by SHWARTZ AND CANTOR in 1984
• High molecular weight DNA with many megabases or even entire chromosomes are separated using Pulsed-Field Gel Electrophoresis (PFGE)
• High molecular weight DNA with many megabases or even entire chromosomes are separated using this technique
• Applications of Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE)

  • Chromosome rearrangement detection
  • RFLP
  • DNA fingerprinting
  • Identifying linked strains in the event of hospital outbreaks, etc.
• Sodium dodecyl sulfate (SDS)


  Detergent in the sample buffer that damages the tertiary structure of proteins by rupturing their disulfide links
• DNA separation in an agarose gel

  Accomplished by changing the direction and strength of the electric field between electrodes
• Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE)


  Method for separating proteins based on polypeptide chain length, largely eliminating the influence of structure and charge
• SDS-PAGE is used to calculate the protein’s molecular weight and determine whether protein samples are pure or not
• Isoelectric point and Isoelectric focusing (IEF)

  pH level where proteins have no net charge (pI), used for high-resolution separation of proteins by their isoelectric points
• 2D-Electrophoresis


  Analyzes complicated protein mixtures by combining 2DGel, IEF, and SDS-PAGE procedures
• Immunoelectrophoresis (Rocket Electrophoresis)

  Separates protein antigen using electrophoresis and immunodiffusion against antiserum to create precipitin
• Difference Gel Electrophoresis (DIGE)

  Addresses the quantitative element of differential-expression investigation
• Difference Gel Electrophoresis (DIGE)

  • Created to address the quantitative element of differential-expression investigations and to alleviate some of the issues with 2D-PAGE, such as analytical fluctuations
  • Up to three different protein samples can be tagged with fluorescent dyes that are size and charge-matched (e.g., Cy3, Cy5, Cy2)
  • Three samples are combined, loaded, and subjected to 2D electrophoresis
• Pipetting


  Technique used in clinical chemistry which involves transferring aliquots of liquid samples from one container to another
• Immuno-precipitates


  Interacts with Ab to form the Ag-Ab complex, and precipitates
• Immuno-precipitates appearance

  • Will then appear as arcs like rockets once the gel has been stained with a suitable dye like CBB
• Week 1: Equipment and Pipetting

  1. Blowout pipette: Last drop of liquid should be expelled into the receiving vessel

  2. Self-draining: Allow contents to be drained by gravity
• Automatic pipettes


  • Pipetting devices that permit rapid repetitive measurement and delivery of precise volumes
  • Two most widely used types: Air displacement, Positive displacement
• Classification of pipette according to type

  • Transfer pipette: Can dispense a known volume of liquid
  • Volumetric pipette: Calibrated to accurately deliver a fixed volume of diluted aqueous solution
  • Ostwald–Folin pipette: Used to accurately measure viscous liquids such as blood
  • Measuring pipette: Which has graduation marks, can dispense a different volume of liquid
  • Mohr pipette: Self-draining and has graduations above the tip
  • Serological pipette: Blowout type and has graduations down the tip
  • Micropipette: Another measuring pipette which can measure less than 1mL