cell fract/protein sep

Question 1

Apoptosis/Necrosis/Annexin V/Propidium Iodide

Answer 1

Apoptosis - cell death by “suicide” - internally programmed cell death
Early apoptosis has a green halo and late apoptosis is red with a green halo
Necrosis - cell death by “murder” from outside influence such as a toxin that causes cell to die
I.e. ricin
Also called pathological cell death
Cell turns red inside during necrosis
Annexin V - used to detect difference due to its ability to bind to phosphatidylserine (inverts during apoptosis)
The cell membrane contains lipids (phosphatidylserine) flips from the inside layer to the outside layer of the membrane. Once it’s flipped, the Annexin V (green dye) can bind to the membrane.
Propidium Iodide - stains nucleus because the membrane is ruptured and therefore the dye can access the interior of the cell

Question 2

Autoradiography

Answer 2

Relies in radioactively tagged molecules to follow or track a particular process
Used less due to problems of disposal and safety
Track DNA synthesis or protein trafficking
DNA synthesis - 3H-thymidine
Can identify dividing cells in breast tumors
Protein trafficking - 35S-methionine
Labels the protein with amino acids and can be tracked down where they go throughout the cell

Question 3

Densitometer

Answer 3

reads optical densities of gels/X-ray film used in autoradiography

Question 4

FISH - Fluorescence In Situ Hybridization

Answer 4

Detect if gene is expressed
Complementary probe molecules that can associate with either DNA or mRNA.
If DNA is to be probed, it has to be destabilized through heat (“melted”) because DNA is double stranded
Denatures the DNA so the FISH probe can match it into the sequence
Once it’s denatured, the FISH probe can come in and bind into the DNA sequence
For mRNA, you don’t need to melt b/c it’s a single stranded sequence
FISH probes can indicate the telomeres of chromosomes
FISH probes bonded to mRNA can be seen in microscopes in different colors from the rest of the cells

Question 5

Intracellular injection techniques

Answer 5

Key to cloning and transfection
Techniques for introducing a molecule of choice into selected living cells for analyzing the function of the molecules in these cells

Question 6

Single cell microinjection using micropipettes

Answer 6

molecules/tracer dye is injected with micropipettes
Can inject DNA, proteins, etc.
Good for one cell at a time (not too common b/c it’s a drawback in many cases)

Question 7

Electroporation

Answer 7

• uses device that can generate transient (short lasting) holes in lots of cells at once
  Membranes reseal and left unharmed
  Molecule of interest passively diffuses into the cell
  Can kill cells easily though

Question 8

Liposomes and nanoparticles - can carry molecules through direct attachment (nanoparticles) or encapsulation (liposomes)

Answer 8

• can carry molecules through direct attachment (nanoparticles) or encapsulation (liposomes)
  Ineffective and carrier molecules can be degraded.
  This process is random meaning that there is a chance it may either escape from the formed endosome or mature into a lysosome.
  Can’t be controlled too much
  DNA can escape from liposome
  Advantage is that viral cells aren’t used

Question 9

Viral transfection

Answer 9

most biologists use it for gene transfection, ineffective, viral genes may end up in nuclear genome
Efficient but run the risk of integrating viral DNA into nuclear DNA sequence

Question 10

SELDI-TOF/MALDI-TOF

Mass spectrometry

Answer 10

SELDI - Surface Enhanced Laser Desorption/ionization
MALDI - Matrix Assisted Laser Desorption/ionization
Advantages is that very small sample amount is necessary - 1 microliter or so
Resolution is one amino acid
Protein Chip arrays - protein profiling and personal medicine
Alzheimer’s protein: Amyloid beta 1-42
Non-Alzheimer’s protein: Amyloid beta 1-40.
Calculating the ratio of the two can be diagnostic of predisposition or disease progression

Question 11

Pulse-Chase

Answer 11

Two-phase technique used to examine cellular processes that take place over a period of time.
During the pulse phase of the experiment, cells are exposed to a labeled compound. The labeled compound is incorporated into the molecule or pathway being studied.
In the chase phase, an unlabeled form replaces the labeled compound. The reaction is monitored to see how long it takes the labeled form of the compound to be replaced by the unlabeled form. There are many ways to label a compound for use in a pulse-chase experiment. Radioisotopes or fluorescent labeling using compounds such as green fluorescent protein (GFP) are both popular.
Watson & crick experiment w/ e coli → DNA is semiconservative

Question 12

Radioactive tagging

Answer 12

Autoradiography uses radio tagging
isotope that is incorporated into a chemical or organic material to allow its detection in metabolic or chemical processes

Question 13

EGTA Protease

Answer 13

How to purify the insulin receptor from single cell type in heterozygous tissue?
Dissociate cells from one another using protease (allows cells to fall apart and be able to locate the proteins) and EGTA - agent that soaks up the calcium that is associated with the protein within the cells
Compound in elution buffer in the protein purification technique known as tandem affinity purification
Lower affinity for magnesium + high selectivity for calcium

Question 14

FACS/ Flow cytometry

Answer 14

FACS- Fluorescence Activated Cell Sorting
Separate cells based on the association of the cell with a specific fluorescent tag
Probe tags cells of interest and not unwanted cells
Operator assigns a voltage to each drop, each drop/cell is deflected into an assigned vial
Voltage allows the machine to choose which cells have the fluorochrome and drop the ones without it into a separate vial (physically separates the cells of interest)
Ex. of application: Apoptosis/Necrosis - Annexin V vs. Propidium Iodide
Guava - $100,000 machine that counts fluorescently tagged cells in multiwell plates but it doesn’t separate them, only counts the number of cells that have a specific color
Can count the number of healthy active mitochondria in cells and the number of inactive mitochondria

Question 15

Dynabeads/Speedbeads

Answer 15

Beads with selective surfaces that can capture cells, proteins, or other molecules based on ligand-receptor interaction
Panning - cell culture based system whereby cells stick if they have the right receptor
Magnetic beads - iron core and ligands on the surface of the bead (can be an antibody) that bind to a receptor on the target cell
Magnet purifies them

Question 16

Veridex

Answer 16

Veridex applications
Veridex CellSearch
Method to detect low number (<1000000000) CTCs
Uses specific antibodies that are tagged with iron (“ferrofluid”) so that it can be pulled out with a magnetic matrix and can be counted by the machine

Question 17

Trituration

Answer 17

Trituration - sucking cells into and out of a small bore needle can sheat the outer cell membrane and release the contents leaving organelles mostly intact (preferred option when it comes to lysing cells)

Question 18

Differential centrifugation

Answer 18

Separate organelles/structures based upon speed of travel through centrifugal field
Rough microsomes (from rough endoplasmic reticulum)
Smooth microsomes (from smooth endoplasmic reticulum, plasma membranes and other organelle membranes
Buffer - physiological saline solution that is isotonic with cells (usually adds protease inhibitors to inhibit protein degradation by lysosomes. Also stabilizes the change in pH

Question 19

Equilibrium Density (rate zonal) centrifugation

Answer 19

particles/molecules travel to a point where their density equals the density of the medium

Question 20

DEAE and CM Ion Exchange chromatography

Answer 20

Ion-exchange chromatography for protein purification
Relies on differences in protein charges
4 types of charged beads (S,CM,Q,DEAE)
DEAE - positive charge, most proteins are negative so they stick to it, and the proteins that don’t stick are released
Sodium chloride gradient - elute proteins (counter-ion)
At neutral pH the CM- cellulose is negatively charged, so it is a weak cation exchanger. DEAE is positively charged at neutral pH and so DEAE-cellulose is a weak anion exchanger.

Question 21

Gel Filtration

Answer 21

Proteins differ in different physical size (molecular weight)
Uses a porous bead so only proteins of a certain size or smaller are able to go through

Question 22

Affinity Chromatography

Answer 22

certain proteins, especially cell membrane receptors such as insulin receptor, bind to other proteins in a specific manner
Covalently cross-linked insulin (ligand) to beads
Add membrane proteins and allow to bind or not bind
Elute bound proteins using high salt concentration
(best choice for isolating the insulin receptor)

Question 23

Protein A immunoprecipitation

Answer 23

Antibodies and protein A (from S. aureus) to purify soluble proteins (centrifuge tube)

Question 24

Dialysis

Answer 24

the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing
Dialysis Diffusion: movement of solutes as a result of random molecular motions across a semipermeable membrane down its concentration gradient

Question 25

Native Gel Electrophoresis

Answer 25

Native gel electrophoresis - based on charge of the protein, physical size, and the extent of crosslinking of the gel matrix, acrylamide
Uses Anode (+) and Cathode (-)
Advantage is that proteins can be removed for further analysis (sequencing or enzymatic activity)
Also retain their native activity

Question 26

SDS gel electrophoresis

Answer 26

Proteins are denatured (no native activity) but resolution is better
Proteins are treated with β-mercaptoethanol, urea and SDS.
Β-mercaptoethanol hydrolyzes disulfide bonds
Urea destroys H-bonds
SDS coats the proteins, giving them equal negative charge/molecular weight basis

Question 27

Isoelectric Focusing

Answer 27

Takes advantage of the fact that each protein has a different pH at which it is electrically neutral - called isoelectric point
Proteins separated on gel where one end has a low pH
Positively charged ones move to cathode
Can resolve proteins of 0.01 pH unit

Question 28

2-D gel electrophoresis

Answer 28

Greatest resolution but most difficult to accomplish properly
Gives more spots, and can differentiate between phosphorylated proteins vs. non-phosphorylated proteins

Question 29

Western blots

Answer 29

Combination of gel electrophoresis and “blotting” technique
Identify a protein based on its ability to bind to an antibody
Both qualitative (indicate molecular weight) and quantitative (indicate amount)
Can determine protein-protein interaction
Relies on UV light and photoreactive amino acids to form a covalent bond b/w ligand and receptor
Western blot can show a band shift to a higher molecular weight indicating that the proteins are covalently bonded
Doesn’t rely on hybridization

Question 30

Cell lines/strains

Answer 30

Cell Line - cells that are immortal/divide infinitely
Embryonic - cell lines derived from embryos
3T3 derived from mouse embryos
Transformed cancer cells - derived from a tumor, transfected with an oncogene(s), exposed to a mutagen
UV light
chemicals
Telomerase immortalized cell lines
Telomerase constantly repairs the telomeres after each division
In cell lines/strain the cells eventually age and due in culture
Cell Strain - cells isolated from normal tissue and placed in culture → can divide a finite amount of times
Leonard Hayflick 1970s: indirect relationship between age of the human donor and the number of potential replications (due to shortening telomeres)

Question 31

Fetal Bovine Serum/defined media

Answer 31

Serum-supplemented media - fetal calf serum that provides necessary growth factors for the cells to survive
Expensive, and introduces unknowns into the culture system
Should never be used to culture stem cells as it might introduce foreign animal (bovine) proteins, prions, viruses
Defined media - all components in media are known, no serum is added

Question 32

Biological Scaffolds

Answer 32

Robert Langer and Joseph Vacanti had led this field in the development of biodegradable scaffolds
Scaffolds are important in developing artificial blood vessels with endothelial cells in the interior
Biodegradable - once cells are seeded, divide and develop the 3-D tissue, the scaffold degrades

Question 33

Tissue Engineering

Answer 33

3-D tissues from cultured cells that will typically form monolayers in vitro
Future of Tissue Engineering:
OB/OB mice - mutant mice are obese
Transplant engineered skin that produced and secreted human leptin - a protein that is known to control food intake
Human leptin (ELISA) was found in serum and found that OB/OB mice ate less
Bioprint 3-D tissue constructs with multiple cell types and “glue them”

Question 34

Microporous Membranes

Answer 34

Cells don’t always exhibit proper phenotype in vitro
MDCK (Madin Darby Canine Kidney) cells must be grown in microporous membrane to demonstrate their appropriate phenotype (1980s)
A growth substrate requirement

Question 35

BioPrinting/3D Printing

Answer 35

additive manufacturing process similar to 3D printing, but unlike 3D printing, bioprinters print with cells and biomaterials, creating organ-like structures that let living cells multiply
allows researchers to fabricate simplified homocellular tissue models for basic research or to produce more complex scaffolds with controlled spatial heterogeneity of physical properties, cellular composition, and ECM/biomolecule organization

Question 36

Decellularization

Answer 36

process used in biomedical engineering to isolate the extracellular matrix of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration
decellularized ECM can then be repopulated with a patient’s own cells to produce a personalized tissue