1. Analysis
1.1. Staining methods
1.1.1. Fluorescent protein labeling Antibodies Negative stain In situ organic dies
1.2. UV/Vis Spectrophotometer
1.2.1. Optic arrangement constituents
1.2.1.1. Source
1.2.1.1.1. Deuterium lap for UV
1.2.1.1.2. Tungsten or halogen lap for visible light
1.2.1.2. Monochromator
1.2.1.2.1. Mechanism that diffracts the light into different wavelenghts in order to select the desired one
1.2.1.3. Container
1.2.1.3.1. cuvette
1.2.1.4. Detector
1.2.1.4.1. Transform the recibed data into electronic data taking advantage onf the electron exitation provoked by the light
1.2.1.5. Signal processor
1.2.1.5.1. Process the transformed datta and make calculations
1.2.1.6. Data readout
1.2.1.6.1. Simplify the data analysis for the scientist
1.3. MALDI and SELDI
1.3.1. MALDI
1.3.1.1. The first step in MALDI is to mix the molecule of interest with the selected matrix. It is important to prepare the sample correctly and to choose an adecuate active ingredient for the matrix, this variables are dependent of the nature of the analyze. A
1.3.2. SELDI
1.3.2.1. Surface-enhanced laser desorption/ionization is a variation of MALDI. SELDI is a soft ionisation technique used in proteomics, it is mainly used for complex protein mixtures. SELDI does not requiere sample pretreatment since it has an affinity surface. Just as in MALDI irradiation by a short laser pulse is required in order to cause ablation and desorption of the sample and matrix. After ionisation MS analysis is applied to the analyte in gas phase using TOF.
2. Stability during storage
2.1. Temperature
2.2. Buffer
2.3. Protein concentration
2.4. Gycerol
3. Recovery
3.1. Liquid-liquid fractionation
3.1.1. Aqueous Two-Phase Systems
3.1.1.1. PEG, Dextran phosphate, sulphate, citrate
3.1.1.1.1. Used to recover proteins, genetic material, bio nanoparticles, cells and organelles
3.1.2. Organic-Aqueous Systems
3.1.2.1. Water ethanol, methanol, butanol, acetone, 2-propanol, acetonitrile
3.1.2.1.1. Protein recovery, but not as efficient as ATPS
3.2. Cell disruption
3.2.1. Bead Mills
3.2.1.1. Bead mills is a mechanical method for cell disruption, cells can be disrupted by being agitated in the presence of small beads, which can be made of various materials
3.2.2. High pressure homogenizer
3.2.2.1. High pressure homogenizer is a mechanical technique used in cell disruption, cells can be disrupted by forcing cells to cross a small orifice through the means of high pressure.
3.2.3. Ultrasonication
3.2.3.1. Ultrasonication or Ultrasonic disintegrators is a mechanical technique for cell disruption, cells can be disrupted by the generation of sonic pressure waves.
3.2.4. Osmotic shock
3.2.4.1. Osmotic Shock is a physical treatment of samples used for cell disruption, cells can be disrupted by causing sudden changes in the concentration of solutes around the cell, this means that concentration of salts in the media must change.
3.2.5. Detergents / Organic solvents / Acids / Bases
3.2.5.1. D/O/A/B are chemical treatments of samples used for cell disruption, these methods rely on cell envelope solubilization or cell membrane reactions for disruption.
3.2.6. Enzymes
3.2.6.1. Enzymes are a biological treatment of samples for cell disruption, cells can be disrupted by three different kind of enzymes: glycosidases responsible for the fragmentation of polysaccharides in peptidoglycan, acetylmuramyl-L-alanine amidases responsible for degradation of polysaccharide-polypeptide junction and endopeptidases responsible of splitting polypeptide chains within peptidoglycan.
4. Purification
4.1. Chromatography
4.1.1. MC
4.1.2. HPLC
4.1.3. Expanded bed chromatography
5. Isolation
5.1. Western blot
5.1.1. Western blot is a technique used for the separation and identification of an specific protein from a complex mix of proteins. This is done in three main steps: Separation by size (done through gel electrophoresis), then the separated protein is transferred onto a solid support (polymer sheet, usually nitrocellulose or PVDF) and finally it is marked using antibodies (incubation with antibodies specific to the protein of interest).