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Technical tools for the study of protein and nucleic acids (Production…
Technical tools for the study of protein and nucleic acids
Sequencing : determination of the primary structure of an unbranched biopolymer
Protein sequencing
From DNA to protein:
Overexpress a candidate DNa to see if we obtain the same protein and if we do find the protein sequence from the DNA sequence
Amino acids analysis
Ion exchange
AAs go through sulfonated matrix based on their isoelectric point and are then labeled with nyhydrin to be detected quantitatively
Reverse phase
AAs are stained with phenylisothiocyate and then run through RP HPLC
Edman's degradation
N terminal AA is selectively cleaved by phenylisothiocyate and the operation is repeated
Making smaller poplypeptides from big proteins
Separation of polypeptide chains and then fragmentation of polypeptide chains using proteases
Mass spectrometry
DNA sequencing
Sanger sequencing : the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
NGS : production of massively parralelised short reads which overlap to some extent allowing reconstruction of the final sequence
Illumina
Nanopore sequencing
SMRT sequencing
Production
Purification from natural sources
De novo chemical production
Heterologous expression in a model organism
Extraction
Purification
Precipitation
Based on solubility
Change of ionic strength of a solution can cause some protein to precipitate but not others
Dialysis
Use of a semi permeable membrane to separate molecules, based on their size
Chromatography :
Size exclusion chromatography (based on size)
Principle : smaller particles can enter the beads (solid phase) and take more time than big particles that by-pass the beads
Technique for mixture separation based on two phases : mobile phase (=liquid carrying the dissolved mixture) will pass through solid phase
Planar chromatography
Column chromatography
Ion exchange chromatography
Based on charge interactions between proteins and solid phase
Affinity chromatography
Based on selective non covalent interactions between analyt and a specific molecule
Adsorbtion chromatography
Electrophoresis
Based on motion of particle in fluid (often polyacrylamide or agarose) under the influence of a uniform electric field
Analyzing the deplacement of a sample in the medium allows to separate molecules based on charge, molecular weight, or shape
Osmotic lysis
Osmotic lysis of cell placed in a hypotonic medium
Based on osmosis = tendancy of a solvent molecules to move through a selectively permeable membrane to equalize the solute concentration
Then : centrifugation to get rid of cellular debris
Mechanical lysis
Cells are disrupted mechanically, by bead grinding, homogeniser, French press, sonicator or freeze-thawing
Detergent-based lysis
Non ionic detergent (+Lysozyme)
