cell structure experiments and diseases

freeze fracture experiment what it is

splits frozen cell membrane to reveal hydrophobic interior of the bilayer

freeze fracture how it is used

helps to see the distribution of integral membrane proteins

dna laddering what it is

assess cell apoptosis by detecting DNA fragments via gel electrophoresis

activated enzymes, specifically caspase-activated DNase, cut genomic DNA

dna laddering what it’s used for

to confirm apoptosis - fragments appear as ladder

differentiation of stem cells in vitro how it works

adds growth factors using signaling molecules to direct cells to differentiation

time-lapse video microscopy what it’s used for

continuous imaging over time to see dynamic processes

Cryo-EM what it

flash freeze samples into an ice layer and then scanning them with electron beams to see 3D structures of delicate proteins and viruses

Cryo EM wat it is used for

helps to visualize biological macromolecules

ultra centrifugation

spinning to separate cellular components by size/density - helps isolate organelles, ribosomes, macromolecules

therapeutic human cloning

somatic cell nuclear transfer into stem cells to show nucleus can be reprogrammed

used for making new tissue to treat diseases to bypass risk of organ rejection

western blot

protein separation via SDS-PAGE then transferring them and using an antibody specificity to detect an antibody-protein complex

used to identify if a protein is present or absent, ex HIV testing

soft agar assay

shows ability of cells to grow without attachment

ames test

uses specific strains that can’t produce histidine, then measuring how often a chemical causes the bacteria to mutate back to a state where they can grow without histidine

used to identify mutagens/carcinogens

PCR

used for DNA amplification for detailed analysis

first denatures DNA to single strands, then uses polymerase to make copies

reverse transcription

RNA to DNA to study gene expression

hierarchy analysis of mutant cells

investigate order and components of a signal transduction pathway

FRAP

bleach flourescent molecules and watch recovery - how quickly the unbleached molecules from surrounding areas move back into that region

shows movement of molecules to measure membrane fluidity and protein mobility

hydropathy plot analysis

graph of hydrophilic vs hydrophobic regions to show potential transmembrane domains and membrane protein topology

sanger sequencing

polymerase extends a strand, and ever so often incorporates a fluorescent ddNTP which prevents further coding because it lacks a 3’oh end

this creates fragments and you can use gel electrophoresis to scan the fluorescense to determine DNA sequence

pyrosequencing

uses light detection of incorporated nucleotides then electrophoresis

also used for DNA sequencing

familial hypercholesterolemia

defects in LDL transport by receptor mediated endocytosis

pulse chase experiment

used to study the movement of cells through organelles

labeled molecule with fluorescence and tracked it through the cell

cloning

need egg cell, donor nuclear material cell, and a surrogate mother to carry the embryo

cancer and rtk pathway

lose of GTPase activity in Ras - ras can’t hydrolyze GTP to GDP and the pathway stays on - constant MAPK signaling

cancer and p53

loss of function of tumor suppressor and damaged cells keep dividing

sickle cell

amino acid substitution - hemoglobin polymerizes under low O2

cystic fibrosis

protein made but misfolded, retained in ER and degraded

neurodevelopmental disorders

defects in cytoskeletal proteins like actin and microtubules

blistering

loss of E-cadherin function - loss of adhesion

more mucus clearance or infertility

cillia mutations - dyenin arm defects