L2Ch18Techniques+toStudyCells2010

Identifying proteins by mass spectrometry
Matrix-Assisted laser Desorption Ionization (MALDI) Electrospray Ionization (ESI)
The result of molecular ionization, ion separation, and ion detection is a spectrum that can provide molecular mass and even structural information
Types of Light Microscopy
Brightfield: Very little contrast. Phase Contrast: Optical contrast: particle retard light by λ. Optics speed light by λ.
European Journal of Human Genetics (2005) 13, 518–519.
Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse Lu et al., Nature Medicine 6 July 2003
NA of best lens is 1.4 Refractive index of immersion oil: 1.515 Refractive index of air: 1 α 1.4 = 1.515 sin a 0.924 = sin a a = 67.53 = half-cone of light 2x = 135o
Sample
MS compatible sample prep. Proteolysis: In-solution In-gel Hybrid Mass spec
Trypsin Column
Analytical HPLC PC station running Search Algorithms Mass spectra
Phase contrast
particle retard light by λ. Optics speed light by λ. λ= destructive interference or constructive interference
As demonstrated by Zernike, the small amount of light diffracted by a microscopic nonabsorbing object still suffers a λ/4 phase shift relative to the undeviated background wave by the very act of being scattered. (The phase shift upon scattering by an absorbing object if λ/2).
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Fluorescence Microscopy
constructs have advantages over siRNA because the effects of these constructs can lead to a more stable and long-term result.
Genomics whole-genome technologies!
Databse Cluster
Reverse Phase Selector
What can you do with Proteomics?
Improving accuracy of annotation Proteomics can provide information that other annotation methods are blind to, such as RNA editing and novel protein modifications. Organ proteome profiling: Analysis of the genes that encode
these proteins might help identify the regulatory regions that direct organ-specific gene expression.
Organelle proteome profiling.
Exon Skipping and Duchenne Muscular Dystrophy
Methods to study cells
Brightfield Microscopy Phase Contrast Microscopy Differential-Interference Microscopy Fluorescence Microscopy 2-photon microscopy Cryoelectron tomography
A deficiency of the membrane protein dystrophin causes the progressive deterioration of muscle fibres in DMD. Sometimes however, DMD patients have rare, dystrophin-positive fibres ('revertant fibres') that originate from exon skipping in the dystrophin gene, which generates a truncated transcript with a restored open reading frame
oligoribonucleotides (AONs).
Antibodies: anticancer therapies RNAi: Gene silencing and knockdown shRNAs: Gene silencing and knockdown. Short hairpin RNA
λ= destructive interference or constructive interference
DIC: optical contrast using polarized light (Polarizer) split into two partial beams by a Wollaston Prism.
Objective Lens: most important single component of scope
Numerical Aperture
NA = η sin α NA: Numerical aperture η : refractive index of medium between object and objective α : half angle of cone of light
MCDB S-205
Lecture #2 Tuesday, 06/01/2010 How Do We Study Cells?
Hale Waihona Puke How was this cell imaged?
Method in Cell and Molecular Biology (Big Science)
Genomics Proteomics Mass Spectroscopy Exon Skipping: safe and efficient systemic delivery of antisense
Widefield Microscopy: Significant of out-of-focus light collected by detector (camera).
Confocal Laser Microscopy: Adjustable pinhole removes out-of-focus light. Optical slice detected by detector (PMT). Enables deep tissue optical sectioning.
Exon Skipping and Duchenne Muscular Dystrophy
AONs vary in length between 16 and 22 nucleotides and are chemically modified to be resistant to intracellular nucleases and RNaseH. It is thought that they bind to specific sequences in the premRNA, and thus disturb exon inclusion signals like splice sites, intronic branch point sequences, or exonic splicing enhancer elements. This, in turn, leads to the removal of the targeted exon from the processed mRNA.
DNA microarrays: tumor typing, diagnostic tool Entered an era of era of personalized medicine. Genetic markers of increased disease risk, Companies that market testing services directly to consumers through the Internet now offer a range of personal genotyping and sequencing services to clients who are interested in probing their genomes 23andMe, deCODE genetics, Navigenics Knome.