Resume
Bio-analyse & farmaceutische toxicologie samenvatting
- Cours
- Établissement
Bio-analyse & farmaceutische toxicologie samenvatting
[Montrer plus]
Vendeur
S'abonner
melisacakir
Aperçu du contenu
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
Les avantages d'acheter des résumés chez Stuvia:
Qualité garantie par les avis des clients
Les clients de Stuvia ont évalués plus de 700 000 résumés. C'est comme ça que vous savez que vous achetez les meilleurs documents.
L’achat facile et rapide
Vous pouvez payer rapidement avec iDeal, carte de crédit ou Stuvia-crédit pour les résumés. Il n'y a pas d'adhésion nécessaire.
Focus sur l’essentiel
Vos camarades écrivent eux-mêmes les notes d’étude, c’est pourquoi les documents sont toujours fiables et à jour. Cela garantit que vous arrivez rapidement au coeur du matériel.
Foire aux questions
Qu'est-ce que j'obtiens en achetant ce document ?
Vous obtenez un PDF, disponible immédiatement après votre achat. Le document acheté est accessible à tout moment, n'importe où et indéfiniment via votre profil.
Garantie de remboursement : comment ça marche ?
Notre garantie de satisfaction garantit que vous trouverez toujours un document d'étude qui vous convient. Vous remplissez un formulaire et notre équipe du service client s'occupe du reste.
Auprès de qui est-ce que j'achète ce résumé ?
Stuvia est une place de marché. Alors, vous n'achetez donc pas ce document chez nous, mais auprès du vendeur melisacakir. Stuvia facilite les paiements au vendeur.
Est-ce que j'aurai un abonnement?
Non, vous n'achetez ce résumé que pour €7,99. Vous n'êtes lié à rien après votre achat.
Peut-on faire confiance à Stuvia ?
4.6 étoiles sur Google & Trustpilot (+1000 avis)
69052 résumés ont été vendus ces 30 derniers jours
Fondée en 2010, la référence pour acheter des résumés depuis déjà 15 ans