Study Guide Terms Exam 1- Cell Separation/Fractionation Flashcards
1
Q
What is EGTA/Protease and how does it work?
A
- 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
2
Q
What research questions do EGTA/Protease attempt to solve?
A
- 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
3
Q
What is FACS and how does it work?
A
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
4
Q
What research questions does FACS address?
A
- 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
5
Q
What is Guava and how does it work?
A
- 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)
6
Q
What research questions does GUAVA attempt to address?
A
- 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
7
Q
What is velocity sedimentation and how does it work?
A
- 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
8
Q
What research questions does velocity sedimentation attempt to address/solve?
A
- seprating blood cells which vary in size/density
- cell differentiation: how do changes in size/density relate to cell differentiation processes?
9
Q
What is panning and how does it work?
A
- surface of well plate coated w specific antibodes/ligands
10
Q
What is panning and how does it work?
A
- 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
11
Q
What reserach questions does panning attempt to answer?
A
- isolating specific cell types based on surface markers
12
Q
What are dyna beads and how do they work?
A
- 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
13
Q
What is Cell search by Veridex and how does it work?
A
- 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
14
Q
What research questions does Cell Search by Veridex try to solve?
A
- monitoring cancer burden over time
- best for following a specific type of cancer CTC number over time, rather than identifying cancer type
15
Q
What is trituration, how does it work, and what reserach q’s does it address?
A
- uses a small syringe to pull cell up and down causing shear stress to separate single organells (w intact membrane)
- used in cell fractionation
16
Q
What is differential centrifugation, how does it work, and what research q’s does it address?
A
- 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
17
Q
What is rate zonal centrifugation, how does it work, and what research q’s does it address?
A
- 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
18
Q
Ion exchange chromatography
A
- 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
19
Q
Gel filtration chromatography
A
- 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
20
Q
Affinity chromatrography
A
- 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
21
Q
liquid chromatography
A
- 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
22
Q
native gel electrophoresis
A
- 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
23
Q
SDS gell electroiphorese
A
- 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
24
Q
Isoelectric focusing
A
- 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
25
2D gel electrophoresis
* 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
26
Western blots
* 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
27
Protein simple wes
automated western blot machine
28
photoreactive amino acids
* 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
29
dialysis
* 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
30
SELDI TOF/MALDI TOF
* 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
31
MALDI-TOF ex.
* 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
32
Protein A immunoprecipitation
* 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
33
autoradiography
* 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
34
densitometry
* weighs proteins from gel electrophoresis
* ctrl set at 1, converts density into relative #s
35
q: What proteins are newly synthesized in response to insulin human skin cells?
* 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
