BIO322 Exam 3 Lecture 14 (Chapter 1 & 4)

examples of invertebrate models

c. elegans, drosophilia

invertebrate models are good bc they

grow fast, cheap, easy to work with

invertebrate models are bad bc they

lack complex organs

examples of vertebrate models

zebrafish, mouse

vertebrate models are good bc they are

similar to humans

vertebrate models are bad bc they are 

expensive, slow to breed

cell culture models/mammalian cells

grow fast, no full tissue/organ interactions

single cell model - eukaryotes

yeast, rapid growth, study cell cycle, transport

single cell model - prokaryotes

e. coli, study molecular biology, protein interaction 

what is a simple light microscope

single convex lens bends light from specimen into viewers eye

the simple light microscope is used for viewing

small thin transparent specimens

what are compound light microscopes

uses light to illuminate specimen, inverted image

compound light microscopes are used to view

small specimens

how do compound light microscopes work

light passes thru condenser/specimen, objective lens magnifies image, ocular lens magnifies image 

robert hook came up with the

modern microscope

total magnification

objective x ocular magnifications

abbe’s equation states that light w/ short wavelength can help you see ____, while lens w/ a higher numerical aperture/better lens capturing more light improves ____

smaller details, clarity

what do you use to see structures smaller than 200 nm?

storm or sim

fluorescence microscopy

shine light, stuff glows, see where things are in cells

interference contact microscopy

splits light into 2 beams, beams recombine/meet, 3d contrast, makes structures and intracellular dynamics visible 

under right conditions, gfp emits

green light

what type of proteins can be made with GFP?

chimeric

chimeric proteins 

2+ proteins fused together 

green fluorescent protein gfp

gfp and protein of interest, protein now glows but same function

to see a specific protein in living cells/insert a fluorescent tag

introduce plasmid under appropriate promoter, edit gene w/ crispr-cas9 to insert tag into cells gene

on which end of the cell’s gene can a fluorescent tag be inserted into?

n or c terminus

dapi/hoechst

bind to dna, stains nucleus blue

phalloidin

fluorescent, binds to actin filaments in cytoskeleton

mitotracker/lysotracker

stain mitochondria/lysosome in live cells 

fm4-64

labels plasma membrane and endocytic vesicles

antibodies are __ shaped

y

what cells produce antibodies?

b cells 

antibodies have __ identical antigen binding sites

2

how to make antibodies in animals/lab

inject animal w/ antigen a, repeated injections stimulate b cells to produce large amounts of anti a antibodies

what are b cells?

white blood cells from immune system 

hybridoma technique/making a monoclonal antibody

obtain antigen, inject antigen into animal, isolate b cells from animal, fuse b cell w/ tumor cell, cell divides forever w/ antibodies of 1 type

fluorescent antibody

detected by fluorescent microscope

gold labeled antibody

detected by e- microscope

western blots detect

protein presence

how do western blots work?

separate by size thru gel electrophoresis, transfer to membrane, use antibodies to find protein

how immunofluorescence assays image proteins in cells

fix cells, permeabilize cells by triton x-100, block non specific binding, add proteins

what does permeabilize cells by triton x-100 mean?

poke holes in membrane to allow antibodies to enter and reach proteins 

live cell imaging w/ fluorescent tags is used to detect

protein movement, cytoskeleton changes, organelle movement, cell divison/shape

advantages of live cell imaging w/ fluorescent tags

see processes in real time, cell stay alive, can be imaged many times

disadvantages of live cell imaging w/ fluorescent tags 

tagging proteins could change protein function/location, needs gene editing, signal issues, can track little proteins at once 

advantages of fixed cell imaging w/ antibodies/immunofluroescence

can see where proteins are inside cell, no gene editing, multiplexing

multiplexing

can label a lot of proteins at once

disadvantages of fixed cell imaging w/ antibodies/immunofluorescence

cant see dynamic processes, fixation can change structures, needs specific antibodies 

fluoresence microscopy uses ___ to visualize molecules, while fluorescence confocal microscopy uses ___ to visualize molecules in 3D

dyes/proteins, lasers/pinholes to block light

how to see something smaller than 200 nm

superresolution microscopy, transmission electron microscopy

superresolution microscopy

localizes fluorescent molecules more precisely, storm

transmission electron microscopy tem

fixed cells dehydrated and embedded in resin, small pieces cut, e- beam passes thru sample, image made

in tem, denser =

darker

cryo-em 

isolate protein complex, prepare sample, use e- to image frozen proteins at high resolution

ways to isolate protein complex

immunoprecipitation, immunoaffinity

what is making a homogenate cell?

breaking open cells to release contents

how to make a homogenate cell

break cell open by high frequency sounds, use mild detergent to make holes in membrane, force contents thru hole w/ pressure, shear cells

immunoprecipitation

using antibody to pull out protein of interest

how to use immunoprecipitation

mix homogenate cell w/ antibody, antibody binds proteins, beads attach to antibody, pull bead and protein out

immunoaffinity chromatography

column based, antibody attaches to a protein, larger samples 

how to do immunoaffinity chromatography

attach antibody to column, put protein mixture thru column, protein of interest sticks to antibody, wash everything else, elute bound protein 

types of chromatography

ion exchange, gel filtration, affinity

in ion exchange chromatography, proteins need to be ____ to be activated

phosphorylated

ion exchange chromatography 

separate charged molecules

ion exchange chromatography uses the principle of

like dissolves like

gel filtration chromatography 

separate molecules by size 

in gel filtration chromatography, the column is full of ___ beads.

porous

in gel filtration, smaller =

longer

in affinity chromatography, __ or__ change makes target let go of beads

ph or salt level

tags used for affinity chromatography

his, gst, mbp, natural ligand/substrate, lectin

his tag

histidines stick to metal ions like Ni or Co

for his tags, when a mixture flows thru a column w/ nickel beads, what type of protein sticks?

his tagged proteins

his tag proteins are released w/

imidazole

gst tag sticks to

glutathione

to release protein from gst, add

more glutathione 

mbp tag binds to

amylose

to release protein from mbp, add

malose

natural ligands are 

natural attractions 

example of natural ligand

atp binding proteins stick to atp agarose

lectin

proteins binding sugars

lectin affinity chromatography

purify glycoproteins, study sugar patterns

e- microscopy 

beams of e- visualize cell structures, not in living cell