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Bio-Analysis & Pharmaceutical Toxicology Summary
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Bio-Analysis & Pharmaceutical Toxicology Summary
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0. Lecture Intro Bioanalyse & FarmatoxSample extraction techniques
Liquid liquid extraction
- Two immiscible solvents are “shaken”, hence a partition of substances according to
their lipophilicity occurs
- i.e. water + dichloromethane
• Voordelen:
- Usable for a wide range of analytes
- Easy
• Nadelen:
- “quick & dirty”
- Process has to be repeated
- Very often micelle formation, sometimes very hard to break
Solid phase extraction
• Voordelen:
- Relatively easy
- Clean extracts
- Good for large sample volumes
• Nadelen:
- “expensive”
- Some experience needed
Andere voorbeelden van Sample extraction techniques
• SPME – solid phase microextraction
• Headspace analysis
• Microdrop extraction
• QuEChERS extraction
• Magnetic beads
Different techniques and principles
• HPLC – high performance liquid chromatography
• (UPLC) – ultra performance/pressure liquid chromatography
• GC – gas chromatography
• CE – capillary electrophoresis
• GE – gel electrophoresis
• (SFC – supercritical fluid chromatography)
• (HTLC – high temperature liquid chromatography)
• (Hydrodynamic and field flow fractionation chromatography)
,1. Gel Electrophoresis Antibodies
Gel electrophoresis
Electrophoresis
• Separation according to mass/charge ration (m/z)
• Separation of a mixture under a applied electrical field
• Moving velocity v, electrical field strength E, electrophoretic movability u are related to each
other according to the following equation (where s = distance, t = time, z = number of
charges per molecule and η= viscosity of the medium)
Sodium Dodecyl Sulfate PolyacrylAmide Gel Electrophoresis (SDS-PAGE)
• Can be used for MW determination
• Gel = polyacrylamide gel, prepared directly before use (NOTE: the monomers are toxic!)
• The pore size of the so prepared gel can be optimized for a specific protein by varying the
amount of crosslinker added
• Solubilise the proteins in a solution containing SDS
• SDS is a negatively charged detergent
• The lipophilic part of the SDS molecule binds to hydrophobic patches of the protein,
thereby unfolding the protein and reducing interactions with lipids or other proteins
• Often Mercaptoethanol is also added
• About 1 SDS molecule per 1.5 to 2 amide bonds is adsorbed
• The proteins are now overall negatively charged
• The proteins are moving through the gel towards the anode (+)
- Small molecules move faster through the pores of the gels, hence a separation on
the protein masses is accomplished
- The molecules are unfolded – no influence of shape etc.
- Small molecules move the longest distance towards the anode
Commassie blue staining
Silver staining
Electrophoresis – isoelectric focusing (IEF)
• The isoelectric point (pI)
- The protein has no netto charge
- Therefore u becomes zero
- No movement, when pH = pI
• 1. unfold protein with mercaptoethanol and i.e urea…
• 2. establish a pH gradient:
- Carrier ampholytes: small molecules containing multiple amino- and carboxylate
groups – narrow spaced pI values. When these molecules are directly included in the
gel, they develop a smooth pH gradient over the entire gel
- Acrylamide buffers – acrylamide derivatives containing carboxylate or tertiary amino
groups
, - Incorporated into the gel at the casting stage
- Covalently bound
2D-Gel Electrophoresis (2D-GE)
• Separation of proteins on basis of charge and size
• Two dimensional protein separation
• First dimension:
- IEF
• separation based on pI values
• Second dimension:
- SDS – PAGE
• Separation based on the molecular weight of the proteins
Protein identification (ID) by MS and 2D gel
• Requires gel spots to be cut out (tedious)
• Ideal for lower to medium throughput (up to 500 spots per day)
• Allows modifications to be detected
• MS allows protein identification by:
- Intact protein molecular weight
- Peptide fingerprint molecular weights
- Sequencing through MS/MS
2D-DIGE (Difference in gel electrophoresis)
• All proteins get a color label
• Visualized on a fluorescence gel scanner
• Proteins of interest are cut from the gel for in-gel digestion and MS analysis
• High protein [C]: bright spot
• Low protein [C]: vague spot
Blotting
• Separation
- Often size-based gel electrophoresis
• Transferring proteins, DNA or RNA onto a carrier membrane
• Immobilization
• Visualization
- Colorant staining
- Autoradiographic visualization of radioactive labelled molecules
- Specific labelling
Blotting
• Southern blot: detection of a specific DNA sequence
• Northern blot: study gene expression by detection of RNA or isolated mRNA
• Western blot: detect specific proteins
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