FINALS LAB

• Proteins are biomolecules composed of a stack of amino acids, forming the building block of the system and performing most of the biological functions of the system
• Protein isolation


  Separating a single type of protein from its source or from mixture of different proteins
• Extraction of protein requires breaking the tissue or cell and immersing it into a solution that can undergo different procedures like freezing, sonication, homogenization, filtration, and permeabilization by an organic solvent
• After the soluble protein has been removed from the insoluble protein, the target protein can be isolated from cell membrane or DNA by means of centrifugation
• Different methods for protein isolation used in the laboratory


  • Chromatography
  • Ultrasound homogenization
  • French press
  • Cryogenic grinding (grinding in liquid nitrogen)
  • Lysis buffer

  
• Extraction of albumin from egg


  1. Place 20mL of egg white in a beaker

  2. Add 2mL of 1M Acetic acid in a dropwise manner. Stir continuously

  3. Filter mixture with cheese cloth

  4. Add 1 ammonium sulphate solution to 1mL of filtrate in a test tube. Stand mixture for 30 minutes

  5. Centrifuge the solution. Place the supernatant in a separate test tube the immerse in an ice bath

  6. Add 50% saturated ammonium sulphate solution to the mixture until turbidity persisted

  7. Stand for 15 minutes. Centrifuge

  8. Discard the supernatant. The precipitate collected is the end product (egg albumin)
• Protein


  Separating a single type of protein from its source or from a mixture of different proteins
• This is important because this can help study the specific protein, its interaction and function with other components of the human body
• Reasons for performing protein extraction


  • To compare the structure of proteins by different organisms
  • To purify a protein in order to identify the gene that encodes it and to resolve proteins by SDS-PAGE
  • Examine an enzyme in a crude extract for physiological studies
  • To study the mechanism of action of an enzyme
  • To diagnose parasitic disease
• Methods for protein isolation


  • Chromatography
  • Ion Exchange Chromatography
  • Gel Filtration Chromatography
  • Chromatography based on hydrophobicity
  • Affinity chromatography
  • Ultrasonic homogenisation
  • French Press
  • Cryogenic grinding
  • Lysis buffer

  
• Ion Exchange Chromatography (IEXC)


  • Different types of proteins are separated based on their net charge
  • One of the most frequently used techniques for protein purification due to its high protein binding capacity
  • Allows elution to take place under mild conditions thereby preserving the normal conformation of the protein sample
  • Its limitations in selectivity remain to be of the greatest disadvantages in using this technique

  
• Chromatography based on hydrophobicity


  • Hydrophobic Interaction Chromatography (HIC) - separation of proteins can be brought about by treating the column with a highly ionic buffer to facilitate the binding of the hydrophilic particles of the protein in the column
  • Reversed Phase Chromatography (RPC) - works by adding organic solvents to the mobile phase to decrease its polarity
• Gel Filtration or Size-Exclusion Chromatography


  • Generally used to separate larger proteins from smaller ones by using a minimal volume of eluate
  • Exhibits good sensitivity and does not lead to sample loss mainly because the solutes do not interact with the stationary phase

  
• Affinity Chromatography


  • Most selective chromatography technique
  • Gives the purest results and is therefore used in completing the protein purification process
  • Since different types of proteins exhibit highly specific interactions with particular ligands under favorable conditions, the target protein can then be adsorbed from the extract as it passes through the column while the other substances will simply be washed away
  • The target can then be eluted and made available for analysis by reversing the prevailing experimental conditions

  
• Ultrasonic Homogenisation


  • Used for tissues like some leaves and a post treatment after grinding
  • Does not require freezing thus may avoid artifacts of freezing but may cause artifacts by heating of sample
• French Press


  • Used for individual cells with or without soft walls
  • Does not require freezing and thus may avoid artifacts of freezing
  • Requires many expensive machinery
• Cryogenic Grinding


  • Used for hard tissues and cells like roots, stems, but also for hard walled cells
  • Low temperature protects the proteins during grinding
  • Time consuming and requires suitable machinery
  • Uses liquid nitrogen
• Lysis Buffer


  • Used for bacteria or animal cells
  • May cause degradation
  • No machinery needed
• SDS-PAGE


  Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - a kind of gel electrophoresis where proteins are separated based on their polypeptide chain lengths with the use of sodium dodecyl sulfate/sodium lauryl sulfate and polyacrylamide gel
• Sodium Lauryl Sulfate


  • A detergent widely used in cleaning products and cosmetics such as shampoo
  • Anionic detergent that binds strongly to protein and causes their denaturation
• Gel Electrophoresis


  One of the laboratory methods for separating DNA, RNA, or protein molecules based on their electric charge or size

  
• The majority of biomolecules exist as electrically charged particles with ionizable functional groups, and a solution containing biomolecules will have either positively or negatively charged ions depending on the pH
• Parts of Gel Electrophoresis


  • Power supply
  • Buffers
  • Support Media
  • Electrophoresis Chamber
  • Container for staining and de-staining gel
  • Electrodes
  • Gel Caster and Comb

  
• Types of Electrophoresis


  • Paper gel electrophoresis
  • Agarose gel electrophoresis
  • Polyacrylamide Gel Electrophoresis (PAGE)
  • Pulse-field gel electrophoresis (PFGE)
  • SDS-PAGE
  • 2D-electrophoresis
  • Immunoelectrophoresis (Rocket Electrophoresis)
  • Difference Gel Electrophoresis (DIGE)

  
• SDS-PAGE


  • Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - Originally called the Laemmli Method
  • Proteins are separated based on polypeptide chain length
  • SDS, a detergent in the sample buffer, and some reducing chemicals work together to damage the tertiary structure of proteins by rupturing their disulfide links
  • Used to calculate the protein's molecular weight and determine whether protein samples are pure or not

  
• SDS is an anionic detergent that binds strongly to protein and causes their denaturation
• Gel Preparation


  • Gels used are vertical slabs, because it is more economical and more sample can be compared with each other when run under identical conditions
  • Gels are prepared in glass containers in which they are to be used. The two glass plates are held together but held apart from each other by plastic spacer, vertical slab gels are run along with the glass plate
  • Choice of percentage of gel to be used depends on the size of the protein sample. Separating gel used may vary from 10% PAGE to 15% PAGE. 15% of gel used for separation of protein having molecular weight 10,000 to 100,000 and 10% of gel used for separation of protein having molecular weight 1,50,000

  
• Sample Application


  1. Dissolved samples can be applied using a micro syringe into wells of the gel

  2. Sample buffer containing 10-15% Sucrose or Glycerol, which increase the density of the buffer and ensures the sinking of the sample in to the wells

  3. Sample buffer contain marker/tracker dye Bromophenol blue. It is a small molecule and it moves freely and indicates the electrophoretic migration

  4. Urea, SDS, Disulfide reducing agent such as ß-Mercaptoethanol are added to protein sample to facilitate their solubilisation

  5. Protein sample can be loaded in the form of a sharp band by using a stacking gel over the separating gel

  6. Only μg of sample are used for analyzing

  
• Running the Gel


  1. The gel slab sandwiched in between the glass plate is placed in the lower reservoir with the top of the gel in contact with the buffer in the upper reservoir

  2. In sample small protein can more easily pass through the pores and larger proteins are successively retarded by frictional resistance due to sieving effect of the gel

  3. Precise voltage and time required for the optimal separation - Voltage: 30 mA; Time: 3 hrs

  
• Detection


  1. When tracker dye reaches the bottom of the gel the current is turned off

  2. Gel slabs are removed without any pressure, after removal gel is immersed in 7% acetic acid to minimize diffusion of components

  3. Then the gel is shaken well in an appropriate stain solution. Usually Coomassie Brilliant Blue R250 is used and the gel is immersed for a few hours

  4. Then the gel is transferred into a destain solution and kept overnight

  5. Destain solution removes unbound background dye from gel leaving stain protein visible as blue bands on a clear background
• Determination of Molecular Weight (Protein) by SDS PAGE


  • The Molecular weight can be determined by comparing mobility of standard protein of known Molecular weight with of unknown Molecular weight that is run on the same gel
  • A calibration curve is constructed for standard protein of known Molecular weight by Distance migrated Vs Molecular weight x 104
  • The migration of unknown is measured are extrapolating this value in the calibration curve, the molecular weight of unknown can be determined
• DNA Sequencing


  • Process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA
  • Sequencing an entire genome (all of an organism's DNA) remains a complex task and requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus"