M1

Isoelektrische Fokussierung, Trennug der Proteine nach pI / bestimmung des pI.

1)Ampholytlösung in Gel einpolymerisieruen

2)Anlegen eines elektrischen Felds --> erzeugt pH-gradient im gel

3) zugabe proteinlösung

4)erneutes anlegen des elektrischen felds

5) (anfärbung) proteine wanderen je pI entlang des pH gradienten

Limitierung:- viele Proteine (gleicher pI), pH außerhalb 3,und 9

-->Trennung nach Molekülmasse

1)Trennung der Proteine durch Isoelektrische Fokussierung im zylindrischen Gel

2)Gel horizontal auf ein zweites, plattenförmiges Gel legen (SDS)

3)Trennung der Proteine mittels SDS-PAGE (SDS-Polyacrylamide-Gelelektrophorese)

-->WesternBlot zur identifizierung (based on antibody binding specifity)

protein identification:

1)cleavage on protein into peptide

2)absolute peptide masses are measured by MALDI-TOF or ESI-TOF

3)compare masses to database or genome

stable associations of sphingolipids and cholestrol, slightly thicker , bilden "liquid-ordered phase" in der fluiden membran. wichitg für signalprozesse, da sich zB GPI-verknüpfte Proteine darin anreichern

..is a branch of proteomics that identifies, catalogs and characterizes proteins containing a phosphate group as a post-translational modification

stable isotope labelling with amino acids in cell culture

-technique based on mann spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labelling (for quantitative proteomics)

1)labelling: grow cell under different conditions (light medium + normal amino acid) (medium + heavy amino acid)

2)metabolic incorperation of amino acids into the protein (mass shift of corresponding peptides, detectd by mass spectrometer)

3) compare both samples--> ratio peak intensity reflects relative protein abundance

-shot gun:Protein-->Peptides-->LC/ESI-->MS-->CDI of random ions-->MS/MS-->database

-targeted:Protein-->Peptides-->LC/ESI-->MS w list of targets--> Q1(specific selection)-->Q2(CDI)-->Q3(MS)-->specific ion pairs, unique for proteins

-reference dara (sequences, studies, metadata,..) -tools+ computational support -protocols/standard operating procedures (SOPs)

National center for Biotechnology Information

-Information, data, tools,..

Basic Local Alignment Tool

-align 2 or more sequneces, large selection of ref.database

online software, automated analysis model

online software, workbench model

easy use of pre-installed software pipeline

large-scale computational analysis independent of local resources

-start at NCBI

-simple & fast analysis first

-figures wherever possible

-often free tools available

23andMe = personal genome test

human samples are tested using targeted sequencing of specific genomic locations based on published research.

Transcriptomics aims to explain genexpression  (RNA-molecules, coding molecules), which genes are actually expressed.

reduced sequencing effort. only coding sequneces are detected (but provides no information about gene expression, just potentially available gene functions)

aims to detect the amount of proteins expressed.

aims to describe the phenotype of a cell "function", quantitative summary of all metabolites under certain conditions.

:-( high technical, bioinformatic and financial efford, large amount of data, high error rate, needs many different and multiple reads

1) random fragmentation of DNA (mechanistical shearing, enzymatic restriciton)

2) sequencing random fragments (overlap phase)

3)bioinformatic assembly of short fragmentes to build genome consensus

"regulation of geneexpression"

"heritable changes in gene expression not attributable to nucleotide sequence variation."

-bisulfite treatment and sequnecing

-restriciton enzyme digenstion

-histone modification (chromatin remodelling)

-DNA methylation (5' methyl C at CpG islands)

einfluss der nahrung auf genexpression

einfluss des genotypes auf die reaktion des organismus auf nahrung

studium der expressionsmuster aller mRNA in einme biolog system

studium des expresionsmusters aller proteine in einem biologischen system