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30 Cards in this Set
- Front
- Back
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Elution power is influenced by 3 factors:
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– Interaction between solvent molecules and
chromatographed compound – Interaction between adsorbed molecules of the mobile phase and the molecules of the sample in the adsorbed phase – Interaction between the adsorbed molecules of the mobile phase and the adsorbent |
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SDS-PAGE sample buffer
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Tris (pH buffer), SDS (sodium dodecyl
sulphate) (anionic detergent), b-mercaptoethanol (breaks disulphide bonds), bromophenol blue dye, glycerol |
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Polyacrylamide gel (PAG) is formed by
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polymerisation of
acrylamide and bis-acrylamide monomers |
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Hapten
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A hapten is a small molecule that can elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.
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4 labels for immunodetection
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horseradish peroxidase, ALP, radioactive Iodine, Fluorescence
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4 fixatives
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10% buffered neutral formalin, ethanol, isopentane, paraformaldehyde
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Viral vectors
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• Exploits natural ability of viruses to infect mammalian
cells • Viruses are genetically engineered to carry and deposit a transgene cassette within a cell • Engineered for a single round of host cell infection by removal of viral genes involved in replication. |
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Developing vectors from viruses
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• Molecular cloning techniques with bacterial plasmid-based
systems used to genetically engineer viruses into vectors • Components of the wild-type viral genome are removed and replaced with a therapeutic gene cassette. The viral packaging signal remains as the one of the cis-acting elements. • The displaced viral genes are placed in a second plasmid(s) - (helper plasmid) or alternatively, are incorporated into a packaging cell line. Helper DNA lacks the packaging domain so it itself or its RNA cannot be packaged into a viral particle. |
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Advantages of viral vectors over transgenic animals for assessing gene expression
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Gene cassettes can be engineered to express one or two
foreign genes, different regulatory elements and promoters Higher levels of transgene expression obtained Vectors can be administered at any developmental stage - from in utero to adult to senescent animals Either short or long-term CNS gene expression Targeted expression in specific tissues, brain regions (sideeffects associated with widespread global overexpression can be avoided) Not host-specific - rats, primates, mice Inexpensive and rapid to generate compared with classical transgenics |
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Non RNAi examples of antisense technology (4)
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phosphodiester oligos, phosphorotioates, peptide nucleic acids, morpholinos
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A mass spectrometer is an
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ion optical device that produces a beam of gas phase ions from samples, sorts these ions according to their mass-to-charge ratios, and determines the intensity (abundance) of each ionic species”.
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Q: What does a mass spectrometer measure?
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A: m/z (mass/charge ratio)
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you might get multiple peaks in mass spectrometry because:
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multiple parent molecules in mixture
fragmentation of parent Isotopes (nuclides) |
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Unified anomic mass units (u)
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= Dalton (Da)
1/12th the mass of a 12C atom at rest in its ground state |
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integer mass
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protons and neutrons of an element
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nominal mass
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the integer masses of the most abundant isotopes of the elements in a compound (CH4 = 16)
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Average mass = molecular weight
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atomic weights of the elements in a compound weighted according to ionic composition CH4 = (12.011+1.008)
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exact mass (monoisotopic mass)
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most abundant isotopes of elements in a compound CH4= (12.000+1.0053)
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Inductively-coupled plasma mass spectrometry (ICP-MS)
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singly charged ions. large differences in sensitivity between ions. most useful for trace metal analysis.
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Accelerator mass spectrometry (AMS)
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measures C13/C14 ratios, extremely sensitive
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Molecular mass spectrometry
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Non-polymers (“small molecules”)
Polymers – especially proteins MW of complete proteins Identification of known proteins in complex mixtures Post-translational modification De novo sequencing Quantitation |
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sample introduction for mass spec
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gases: GC/MS
liquids: HPLC, Flow injection analysis, capillary electrophoresis Solids: matrix-assisted laser desorption ionisation |
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Electron ionisation (EI)
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volatile and thermally stable compounds only, large libraries, well understood fragmentation patterns, often no molecular ion.
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Atmospheric pressure chemical ionisation
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Cmpds of low/moderate polarity
<1.5 kDa Less fragmentation than EI Easier to identify molecular ion |
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Electrospray ionisation (ESI)
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Polar molecules
Includes fagile biomolecules of high MW Least fragmentation Best method for finding molecular ion |
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Matrix-Assisted Laser Desorption Ionisation (MALDI)
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Very important for non-volatile analytes, especially proteins.
Multiple samples on single plate allows rapid analysis Tends to give singly-charged species |
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ion traps
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Easy to use, small footprint, low cost. Excellent for structural elucidation. Sensitivity/quantitative performance not quite as good as triple quad.
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Proteomics applications of MS
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Determination of MW
Protein identification - De novo sequencing - Database matching Post-translational modification Changes in expression (relative quantitation) |
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DECONVOLUTION: (proteomics mass spec)
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Mathematical transformation of a spectrum of several multiply charged peaks into one peak corresponding to a singly charged ion.
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SILAC
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Stable Isotope Labelling with Amino acids in Cell culture
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