Study Guide Terms Exam 1- Cell Separation/Fractionation

Question 1

What is EGTA/Protease and how does it work?

Answer 1

• EGTA: calcium chelating agent that removes Ca2+ ions from the extracellular environment, which compromises Ca2+ dependent cell adhesion molecules
• Protease: (trypsin-research, liberase-cell therapy) cleaves proteins bc other proteins are holding cells together

used in cell dissociation

Question 2

What research questions do EGTA/Protease attempt to solve?

Answer 2

• allows for gentle dissociation of cells including cells from tissues so they can be used for single cell analysis or further culture
• cell isolation for downstream applications

Question 3

What is FACS and how does it work?

Answer 3

Fluorescence activated cell sorting, flow cytometry
* cells labeled w fluroscent mAb that bind to (recognize) cell of interest- fluorochromes different depending on cell receptors
* labeled cells are passed through a narrow nozle and flow through a laser beam (to excite), and emitted light is detected by a series of detectors
* detectors measure the intensity of fluorescence for each marker and apply an electric charge
* charged molecules are sorted based on charge- sorting cells ito distinct populations

Question 4

What research questions does FACS address?

Answer 4

• Sort cells into groups based on specific markers (ex. stem cells, cancer cells, immune cells)
• Sorts individual cells, allowing for highly accurate isolation of specific cell types or subpopulations

Question 5

What is Guava and how does it work?

Answer 5

• Offers flow cytometry capabilites for analyzing fluorescently tagged cells using microcapillary tubing (doesn’t sort, just counts)
• Flow analyzer- not cell sorter (do not physically separate cells into different populations like FACS)

Question 6

What research questions does GUAVA attempt to address?

Answer 6

• analyzing data from cell samples to provide measurements like: cell count, viability, proliferation
• how many cells are healthy vs sick?
• quantifying immune cell subtypes in response to various things

Question 7

What is velocity sedimentation and how does it work?

Answer 7

• sorts cells based on size/density by allowing them to settle through a Ficoll solution at different rates
• separates purely by physical properties (no fluorescence)
• larger/denser cells move faster through solution and are collected first

Question 8

What research questions does velocity sedimentation attempt to address/solve?

Answer 8

• seprating blood cells which vary in size/density
• cell differentiation: how do changes in size/density relate to cell differentiation processes?

Question 9

What is panning and how does it work?

Answer 9

• surface of well plate coated w specific antibodes/ligands

Question 10

What is panning and how does it work?

Answer 10

• well plate coated w specific antibodies/ligands that bind to cell surface markers unique to the target cell type
• cells placed into coated dish- those that have the right receptor will bind to the coating, the ones that don’t will remain unattached
• dish washed to remove unbound cells

Question 11

What reserach questions does panning attempt to answer?

Answer 11

• isolating specific cell types based on surface markers

Question 12

What are dyna beads and how do they work?

Answer 12

• tiny magnetic beads coated w antibodies that bind to specific cell surface markers, allowing for the targeted isolation of cells from a mixed population using a magnetic field
• magnet draws in cells that express target marker

good for isolating particular cell types from mixed sample, often used for capturing CTCs

Question 13

What is Cell search by Veridex and how does it work?

Answer 13

• FDA approved for counting rare CTCs (as low as 1 cell/billion) and analyzing tumor burden over time
• blood sample is mixed with magnetic nanoparticles coated w antibodies specific for EpCAM
• antibodies bind to the CTCs (EpCAM is almost unique CTC cell surface protein)
• then counts CTCs using magnetic plates

Question 14

What research questions does Cell Search by Veridex try to solve?

Answer 14

• monitoring cancer burden over time
• best for following a specific type of cancer CTC number over time, rather than identifying cancer type

Question 15

What is trituration, how does it work, and what reserach q’s does it address?

Answer 15

• uses a small syringe to pull cell up and down causing shear stress to separate single organells (w intact membrane)
• used in cell fractionation

Question 16

What is differential centrifugation, how does it work, and what research q’s does it address?

Answer 16

• loose organells from trituration in isotonic solution with buffer are spun
• nuclei separate and the rest is spun harder
• mitochondria,peroxisomes, lysosomes separate out, spin the rest again harder
• microsome fraction separates out

used to isolate organelles for for biochemical analysis

Question 17

What is rate zonal centrifugation, how does it work, and what research q’s does it address?

Answer 17

• rate zonal=equilibrium
• density=density gradient (using Ficoll)
• organelle reaches a point of equal density w ficoll (smooth microsomes on top, rough on bottom)
• used to separate microsome fraction into rough and smooth

Question 18

Ion exchange chromatography

Answer 18

• good starting point if you don’t know a lot about your protein
• charged beads (DEAE+/CM-)
• sample applied to column- molecules w opposite charge to the resin bind, allows you to separate protein fractions (proteins are - charged)

NaCl gradient as counter ion

Question 19

Gel filtration chromatography

Answer 19

• sample moves through gell w pores
• large molecules too large to enter pores and travel quickly
• small molecules enter pores and take longest path, coming out last
• used to separate proteins based on size

Question 20

Affinity chromatrography

Answer 20

• how insulin receptor was purified for the 1st time
• separates proteins based on interactions w POI
• molecules loaded onto column containing ligand that the POI can bind to
• proteins bind, the rest are washed out
• Nacl is added so receptors come off
• use desalting colmn to remove sodium chloride

Question 21

liquid chromatography

Answer 21

• mobile and stationary phase
• small amount of sample injected into column and components interact differently w the stationary and mobile plases, leading to their separation
• separation based on polarity, size, charge, or hydrophobicity

Question 22

native gel electrophoresis

Answer 22

• gel (polyacrylamie)
• proteins in native state loaded into gel and subjected to an electric field
• bc in native form protein migration influenced by weight (HMW on top, LMW on bottom) and net charge

Question 23

SDS gell electroiphorese

Answer 23

• better resolution than native, multimeric protens turned into monomers and visualized in gel
• boil proteins and add urea to disrupt H bonds
• add beta-mercaptoethanol to hydrolyze disulfide
• SDS to coat the protein w negative charges

Question 24

Isoelectric focusing

Answer 24

• pH gradient in gel
• proteins migrate until they reach their isolectric point (point in pH gradient where net chare is zero), leads to separation based on charge differences
• tightness of bands better than sps

eg blood serum has 40 diff bands

Question 25

2D gel electrophoresis

Answer 25

• best resolution of all 4
• first proteins separated by isoelectric focusing
• then proteins separated using SDS
• can distinguish phosphorlated proteins from non phosphoralated parents
• can cut out piece of gelatin and do SELDI or MALDI-TOF

Question 26

Western blots

Answer 26

• gel eelctrophoresis and a blotting paper
• mAbs to identify POI (can get MW-qualitative, and compare amounts-quantitative)
• draw proteins out of gel onto transfer paper w electric current
• add mAbs to determine band with POI

Question 27

Protein simple wes

Answer 27

automated western blot machine

Question 28

photoreactive amino acids

Answer 28

• amino acids that respond to ultraviolet light and as a consequence create covalent bonds
• binds to POI, add UV light and cannot bind
• THEN do western blot- w UV light can see what POI binds to

tracking ligand receptor interactions in living cells

Question 29

dialysis

Answer 29

• small molecules and buffer pass through membrane in bage, while larger molecules retained in bag
• reduces concentration of small molecules in sample so it can be exchanged into a different buffer system

Question 30

SELDI TOF/MALDI TOF

Answer 30

• surface enhanced laser desorption ionization/matrix associate laser desorption ionization (time of flight)
• characterize proteins and have resolution of 1 amino aci
• components: laser, ionized protein sample, protein chip (chromographic surface w +/- or mAb to keep protein stuck), TOF detector determines how long it takes for the proteins to leave the chip and land on plate

beasts all others in terms of resolution

Question 31

MALDI-TOF ex.

Answer 31

• beta amyloid (has many molecular weights)
• 42=42 AA=shows dementia
• 40=40AA-normal
• chip has mAb that binds to all betaamyloid fragments
• can tell if someone has more 42 and is predementia

Question 32

Protein A immunoprecipitation

Answer 32

• protein A-isolated S. aureus will bind to all mAbs
• inert bead modified to have protein A
• bead added to solution of mixed proteins and mAbs
• mAbs which are bound to POI now bind to bead’s protein A
• centrifuge to separate pellets

Question 33

autoradiography

Answer 33

• radioactive probe to monitor a cells activity
• gel electrophoresis
• add probe, do electrophoresis, analyze to detect radioactive bands
• S-35 methionin probe for newly synthesized proteins, P-32 for phosphorylated proteins

Question 34

densitometry

Answer 34

• weighs proteins from gel electrophoresis
• ctrl set at 1, converts density into relative #s

Question 35

q: What proteins are newly synthesized in response to insulin human skin cells?

Answer 35

• pick probe (S-35)
• control (no insulin) given S-35 methionine
• experimental group given insulin and s-35 meth
• SDS electrophoresis and autoradiography done to both
• both have bands, but bands present in only experimental sample show those that were made in response to insulin