MolBio

• Proteins


  Biomolecules composed of stacks of amino acids, forming the building blocks of the system and performing most of the biological functions
• Protein Isolation


  Separating a single type of protein from its source or from a mixture of different proteins
• Importance of Protein Isolation


  • It can help us study the specific protein, its interactions and function with other components of the human body
• Reasons for Protein Isolation


  • 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
• Ion exchange chromatography (IEXC)


  Different types of proteins are separated based on their net charge (facilitates both anion exchange and cation exchange)
• Ion exchange chromatography (IEXC)


  • 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 one of the greatest disadvantages in using this technique
• 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
• Hydrophobic Interaction Chromatography


  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
• Affinity Chromatography


  • Most selective chromatography technique
  • Gives the purest results and is therefore used in completing the protein purification process
  • 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 artefacts of freezing but may cause artefacts by heating of sample
• French Press


  • Used for individual cells with or without soft walls
  • Does not require freezing and thus may avoid artefacts 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
• 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
• 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 Gel Electrophoresis


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

  
• SDS-PAGE


  • Originally called the Laemmli Method after its British inventor U.K. Laemmli
  • Upper stacking gel has larger pores with a pH of 6.8, and Lower Separating Gel has smaller pores with a pH of 8
  • Proteins are separated based on polypeptide chain length in SDS-PAGE, which largely eliminates the influence of the structure and charge thanks to the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel
  • 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
  • It is used to calculate the protein's molecular weight and determine whether protein samples are pure or not

  
• Principle of SDS-PAGE

  SDS is an anionic detergent that binds strongly to protein and causes their Denaturation
• Gel Preparation for SDS-PAGE


  • Gels used are vertical slabs, because it is more economical and more sample can be compared with each other when run under identical conditions
  • Choice of percentage of gel to be used depends on the size of the protein sample
  • 15 % of gel used for separation of protein having molecular weight 10,000 to 1,000,000
  • 10 % of gel used for separation of protein having molecular weight 1,500,000
• Sample Application for SDS-PAGE


  • Dissolved samples can be applied using a micro syringe into wells of the gel
  • Sample buffer containing 10-15 % Sucrose or Glycerol, which increases the density of the buffer and ensures the sinking of the sample into the wells
  • Sample buffers contain marker/tracker dye Bromophenol blue
  • Urea, SDS, Disulfide reducing agents such as β-Mercaptoethanol are added to protein sample to facilitate their solubilisation
  • Only μ g of samples are used for analyzing

  
• Running the SDS-PAGE Gel


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

  
• Detection of Proteins in SDS-PAGE

  • When tracker dye reaches the bottom of the gel the current is turned off
  • Gel slabs are removed without any pressure, after removal gel is immersed in 7 % acetic acid to minimize diffusion of components
  • Then the gel is shaken well in an appropriate stain solution, usually Commassive Brilliant Blue R250
  • Then the gel is transferred in to a destain solution and kept for overnight to remove unbound background dye from gel leaving stain protein visible as blue bands on a clear background
• Determination of Molecular Weight 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 by extrapolating this value in the calibration curve, the molecular weight of unknown can be determined
• Why do we sequence DNA/ genome?

  
• Maxam-Gilbert Chemical Cleavage Method


  • Developed by Alan Maxam and Walter Gilbert
  • Radioactive labeling at 5' end of the DNA fragment
  • Chemical cleaving in sequence-dependent manner

  
• Sanger Chain Termination Method


  • Developed by Frederick Sanger
  • Enzymatic termination of DNA synthesis at random sites using dideoxynucleotides
  • For routine sequencing applications
• Reagents for Sanger Sequencing


  • ssDNA as template
  • Oligonucleotide primers
  • DNA polymerase
  • Dideoxynucleotide triphosphates (ddNTPs)

  
• Sanger Sequencing Method


  • Fragments are separated in 4 lanes
  • Visualized by UV light
  • Development of automated sequencer
  • Uses fluorescent tags, one for each base: Adenine, Guanine, Thymine, Cytosine
  • Sequence is recorded as chromatogram

  
• Next Generation Sequencing (NGS) Technologies


  • Enabled sequencing of millions of DNA molecules simultaneously
  • Can be used in entire genome sequencing
  • High- throughput

  
• Pyrosequencing (Roche 454)


  • Relies on the detection of pyrophosphate
  • dsDNA are denatured into ssDNA and are captured by beads
  • Amplification by Emulsion PCR
  • dNTPs complement the template bases
  • PPi is released as bases are added
  • PPi transformed to ATP
  • Measured by chemiluminescence

  
• Sequencing by synthesis (Illumina-Solexa)


  • DNA shearing and adapter ligation
  • Graaft into flow cell or single molecular array
  • Bridge amplification to form clonal DNA fragments
  • Sequencing using ddNTPs with cleavable fluorescent dyes

  
• Sequencing by Ligation (Solid-Applied Biosystems)


  • Sequencing by Oligo Ligation Detection
  • DNA is sheared and adapters are ligated
  • Hybridization to beads
  • Amplification by emPCR
  • Beads attached to glass slides
  • Fluorescent dye-labeled probes for sequencing

  
• Iontorrent Semiconductor Sequencing (Life Technologies)


  • Hydrogen ions are released as nucleotides are added
  • pH change detected by ion sensor
• Real-Time Long-Read Sequencing (Oxford Nanopore Technologies)


  • DNA is initially fragmented to 8–10 kb
  • A leader and a hairpin adapters are ligated to either end of the fragmented dsDNA
  • Adapters direct dsDNA through the pore
  • Voltage shifts as DNA passes through the pore