Exam 1 Flashcards
Name 4 main cell isolation methods
Tissue laser microdisection, immunoprecipitaion, differential centerfugation, flow cytometry with FACS
FACS stands for
Florescence Activated Cell Sorting
Flow Cytometry and FACS steps
cells are florescently labeled, suspended in individual droplets, ran through a laser and identified
+FACS, electrical charge is applied to the cells based on type, then used to sort into the appropriate tube
Mass spec uses
used to identify protein expression levels and modifications
Mass Spec types
- Standard
2. Tandem
Mass Spec process
ion charge is applied in a vacuum, protein is fragmented, fragments are displayed at each mass
Protein analysis by ChIP is used
Used to detect protein bound to regulatory sequences
ChIP steps
- protein fixed to DNA with formaldehyde
- cell is lysed
- DNA is fragmented with sound waves or enzymes
- precipitation using antibodies bind target proteins, and thus also the bound DNA
- amplify precipitated DNA via PCR
SH2
domain that binds phos Tyrosine residues
SH2 domain is important for
cell signaling
found on adaptor proteins, signal transduction
Src kinase has – domains
4
Src kinase 4 domains do what?
2 - kinase activity
1 sh2 - recognize phospho tyrosine
1 sh3 - bind to proline rich peptides for signal transduction
domain evolution driven by -
domain shuffling and gene duplication
greater number of non covalent interactions — how well ligand will bind in binding site
increases
Km is a measure of
conc at 1/2 Vmax, shows enzyme substrate affinity, lower = higher affinity
2 ways phosphate groups can be used to regulate protein function
- addition of P to OH group directly, reversible, can turn a protein on or off
ex. activation of Src kinase - GTP binding proteins, P is attached or not to GTP, instead of directly to the protein
Src kinase activation
- removal of P to loosen conf,
- activating ligand binds to SH3 domain
- kinase phos tyrosine to self activate
in lipid membrane, higher cis bonds =
greater fluidity
3 types of transport that require no energy
- passive diffusion
- facilitated diffusion
- filtration
2 types of transport that require energy
- pinocytosis
2. active transport
types of drug transport depend upon
size, plolarity and hydrophobicity of the drug
water soluble drugs can pass via
passive diffusion through aqueous channels
lipid soluble drugs can travel via
hydrophobic binding with the lipid membrane = passive diffusion
non soluble and non lipid drugs must pass through membrane using
active transport OR facilitated diffusion
most drugs are —
electrolytes (weak/acids or bases)
orally administered drugs must pass though
- epithelium - must cross in and back out of these cells
2. endothelium - loosely packed lining of capillaries, can diffuse in between the cells passively
Blood brain barrier is a result of
tightly packed endothelium of capillaries, prevents passive diffusion
— of drugs cannot cross into brain
98%
3 ways to circumvent blood brain barrier
- highly lipophilic drugs can diffuse through
- specialized transporters can be used
- drug may be injected directly into the CSF
way to get molecule into the brain?
make it lipophilic
Mutant CFTR allele can cause (2)
- less of the protein to be produces OR 2. a less effective variant of the protein to be produced
CFTR defect CLASS I
no synthesis of the protein occurs
CFTR defect CLASS II
protein processing issue, not folded correctly
CFTR defect CLASS III
ion gating defect reduced channel opening
CFTR defect CLASS IV
reduced channel conductance, so ion can’t pass through as easily
CFTR defect CLASS V
splicing defect results in lowered synthesis
CFTR defect CLASS VI
protein instability leads to higher turnover rate on the membrane
CFTR defects classes one, two, five and six result in
reduced quality of the transporter
CRTR defects three and four result in
reduced function of CFTR at the surface
