Extraction and Purification of nucleic acids-Proteins Flashcards
Is the total set of proteins encoded by genome
Proteome
Proteins can be extracted from
various sources, including:
Microorganism
Animal cell or tissue
Separates soluble proteins from cell debris
Centrifiguration
Removes particulates and aggregates
Filtration
Exploits solubility differences
Salting out (Ammonium sulfate precipitation)
Uses pH to precipitate proteins at their isoelectric point.
Isoelectric precipitation
Separates proteins based on their size
Size exclusion chromatography
Separates proteins based on charge
Ion exchange chromatography
Uses a ligand that specifically binds to the target protein.
Affinity Chromatography
Exploits hydrophobicity differences
Hydrophobic interaction chromatography
Protocols are generally employed when the sample of interest consist of cells that lyse easily, such as red blood cells and tissue culture cells
Gentle cell distruption
Cell swell and burst when they are suspended in hypotonic solution, releasing all cellular components.
Osmotic lysis
cells can be lysed by subjecting them to one or more cycles of quick freezing using liquid nitrogen and subsequent thawing
Freeze thaw lysis
cellular membranes can be
solubilized just by suspending the cells in many detergent-containing solutions.
Detergent lysis
by enzymes that specifically digest the cell wall.
,Isosmotic solution
What are the enzyme present in the isosmotic solution
Cellulase
Pectinase for plant cells
Lyticase for yeast cells
Lysozyme for bacterial cells
Biological material with tough cell walls and many tissue types require more rigorous methods, which are mainly based on mechanical rupture.
Sonication
French press
mortar and pestle
Mechanical homogenization
A cell suspension, cooled on ice to avoid heating, can be distrupted by shear forces using short burst of ultrasonic waves
Sonication
Cells are lysed by shear force by forcing a cell suspension through a small orifice at high pressure
French press
Breaking down cells of solid tissues and microorganisms.
Mortar and pestle
Soft, solid, tissues can be employed using handheld devices, blenders, or other motorized devices
Mechanical Homogenization
Can also be employed for cell distruption.
Glass bead homogenization
The uses of precipitation vary based on three (3) main
factors:
Detergent of contanimants for removal
Native or denatured state of proteins
Post processing analysis
This precipitation method is a
concentration-based procedure.
Acetone precipitation
Protein solution is mixed with a dilute sodium deoxycholate buffer
TCA (Trichloroacetic acid) Precipitation
precipitation is remarkably efficient,
even in dilute samples, with recovery values from 90% to 100% of total protein.
TCA-DOC
method of precipitation and
concentration for dilute samples, especially those containing membrane proteins.
Methanol chloroform precipitation
The ratio of
methanol:chloroform: water is
4:1:3
Analyzes protein
purity and molecular weight
SDS PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Identifies proteins using antibodies
Western blot
Determines protein mass and structure
Mass spectrometry
measures protein concentration
UV-VIS Spectroscopy (280 Nm)
Assesses functionality if the protein is an enzyme
Enzyme activity assays
