Cell/molecular Biology Exam 1 - Techniques

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How can cells be isolated and grown outside of an organism?

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- Can isolate and culture cells of your choosing
- You can break open the cells to get the contents
- you can isolate various cellular components and proteins
- you can separate the contents further with SDS-PAGE & 2-D Gel Electrophoresis
- you can ID proteins if you know what to look for (Western blots) or even if you don't (Mass spec)

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Ion exchange chromatography

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charged beads in the column collect oppositely charged particles, the "caught" particles will move slower through the column and allow for separation

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Gel filtration chromatography

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beads in the column contain porous holes which catch smaller components in the sample, causing these smaller components to pass through at a slower rate, separating them from the larger components

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Affinity Chromatography

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beads in the column are covalently attached to a substrate that certain molecules in the sample are able to attach to... the rest pass through, thus separating the components

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SDS-PAGE

(sodium dodecyl sulfate-polyacrylanide gel electrophoresis)

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Proteins are composed of polypeptide chains that fold.
* Different amino acids have different charges which cause them to move differently when placed in an electric field.
* Size also affects the way they move.

--- SDS binds to the hydrophobic regions of the proteins, causing them to denature.
--- If you add the sample onto a gel electrophoresis, this will separate the molecules in the sample by charge and size.

* After SDS PAGE, you can...
... stain the gel to see the protein with a "reversible" stain
... isolate the desired protein by cutting them out with a razor blade
... perform a western blot on the sample

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Western Blotting

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** Use to find out if the desired protein was isolated

- have a gel with sample on it after gel electrophoresis
- use a porous membrane sheet to touch the gel
- use an electric current to get the proteins stuck on the membrane & off the gel
- throw the gel away & put the membrane on a tray
- add antibodies that specifically bind to the desired protein - PRIMARY ANTIBODY
- wash the membrane so all other proteins and materials wash away, leaving only desired protein & primary antibody
- use a SECONDARY ANTIBODY that is specific to the primary antibody
secondary antibody has a REPORTER ENZYME
- wash away any excess secondary antibody that is not bound
- add color detection reagent which is a substrate for secondary antibody -> thus, anywhere that has secondary antibody, primary antibody & desired protein will show color

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Two-dimensional gel electrophoresis

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* based on size & isoelectric focusing

- proteins have...
... carboxylic acid groups (COOH/COO-)
... amine groups (NH3+/NH2)

** Isoelectric focusing is based on the pH (hydrogen content)
-- high pH = protonated/ low pH = deprotonated

- ISOELECTRIC PH POINT: where the protein has no net charge because the +/- charges balance... it will no longer migrate in the electric field

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Mass Spectrometry

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-- peptides are mixed with organix acids and dried onto a slide
-- a laser blasts the sample into ionized gas
-- ionized peptides accelerate into electric field and fly towards a detector
-- how long it takes to get to the detector depends on the mass an charge
** will be signature peaks for different proteins, thus you are able to identify the protein

** Good technique if you don't know what you are looking for

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Co-immunoprecipitation


(PROTEIN-PROTEIN INTERACTION)

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** used to isolate & concentrate a particular protein from a sample that contains many types of proteins

-- beads containing a receptor that is able to bind to a specific protein when there is an antibody that is attached to it
-- antibody specific for desired protein binds to it and then binds to the receptor
-- the beads are run through an SDS PAGE to separate components of the mixture
-- gel is analyzed with Western Blots to confirm the presence of the desired protein

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High Density Protein Array


(PROTEIN-PROTEIN INTERACTION)

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-- spot a tray will thousands of different proteins, keeping track of where you put each spot
-- incubate the tray with the desired protein binder
-- if the protein binds to say spot #2057, you go back and find which protein this is, this is the target protein

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Surface plasmon resonance


(PROTEIN-PROTEIN INTERACTION)

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** must have an idea of suspect proteins




-- light beam passes through a prism at a "resonance angle"
-- energy from the light hits a cloud of electrons at different angles, creating a PLASMON and generating an electric field
-- pass through certain prey molecules to see if they bind to the bait, which causes a disturbance

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FRET (fluorescence resonance energy transfer)


(PROTEIN-PROTEIN INTERACTION)

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-- attach a protein to a fluoresent protein
-- pass light through at a specific wavelength, which is then emitted at a different wavelength

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Methods for Introducing Membrane-Impermeable Substances into Cells:

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1. use a glass micropipette to microinject the substance into the cell

2. cell is placed in a solution of the substance and then two electrodes are placed at either side with give a very short electric shock where transient pores in the membrane allow for the substance to enter the cell

3. membrane-enclosed vesicles that contain the desired substance... they fuse with the plasma membrane and substance is released into the cell cytoplasm

4. gold particles coated with DNA... shot into the cell at high velocity

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Wild Type Gene

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normal version of the gene

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NMR (Nuclear magnetic resonance)

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-- sample is analyzed by looking at the electron spin in a magnetic field
-- movement/vibrations of certain bonds between atoms absorb at differing frequencies, and can be determines by the NMR of the molecule
-- involved peaks that are "upfield"/"downfield" and triplets, doublets, etc. tell the "neighbors"

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X-ray crystallography

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In order to purify a protein using this method, it must be able to crystallize.
-- once it forms its crystalline structure, you shine a light (x-ray) on it and analyze the diffraction of the light.
-- helps to determine the amino acid


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Three ways to enrich a tissue sample for cell isolation:

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1. FACS


2. Magnetized Beads


3. Microdissection

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FACS (fluoresence-activated cell sorter)

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-- antibodies bind to the antigens
-- incubate the cells with a cell antibody that is specific to the desired cell
-- the antibody is coupled with fluorescent materials
-- this passes through a laser and then gets filtered, leaving desired isolated cells

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Magnetized Beads

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-- beads are coupled with antibodies that are specific to the desired cells
-- desired cells bind to these and magnetic beads are passed near a magnet
-- rest of material is washed away and you are left with the cells you want



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Microdissection

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-- place the tissue on a plate and use a laser to cut out the desired tissues/cells

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What three things to cells need in order to grow?

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1. Attachment - a medium to grow on, such as a petri dish - you can sometimes coat the surface with extracellular proteins to facilitate growing

2. Nutrients & Serum - nutrients = cell food
serum = for growth and mitogen

3. The right conditions - Temperature, pH, Humidity... they all must be correct for certain cells to survive and thrive.

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Primary Cells

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Cells taken out of a live organism
- most will not grow forever in a lab, they will die outside of their natural environment

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How to we turn primary cells into cell lines?

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infect the primary cells with certain viruses

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Cell Lines

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Groups of cells established as a line
- these cells can grow forever in the lab with proper care
- when the cells run out of room for growth, a little is taken out in order to start a new colony (PASSAGING/SPLITTING)
- example of a cell line = HELA CELLS

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Passaging or Splitting

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when the cells run out of room for growth, a little is taken out in order to start a new colony

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The two steps in purifying proteins:

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1. Break open the cells
2. Separate the proteins

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How does one break open the protein cells in order to purify them?

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can use:
- blenders
- ultrasound vibration
- osmotic shock (disturb osmosis to burst cells)

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What are the three methods of separation proteins for analysis?

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1. Centrifugation
2. Density Gradient:
3. Column Chromatography

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What four techniques can be used to analyze the purified proteins?

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1. SDS PAGE
2. Western Blotting
3. Mass Spectrometry
4. 2-D Separation (2-D Gel Electrophoresis)

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Two-Hybrid Technique

(PROTEIN-PROTEIN INTERACTION)

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-- make a library for all the proteins you would like to screen for

(library = a collection of bacteria/viruses/yeast where each expresses something different - like a different protein)







-- protein couples to the bait, turns on a gene for the enzyme that allows the yeast to survive... if the yeast survived, then the binding partner worked & can be used to identify which protein worked

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Protein Affinity Chromatography


(PROTEIN-PROTEIN INTERACTION)

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-- see picture








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How to you create recombinant DNA molecules?

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by splicing together two or more DNA fragments

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Phenotype

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appearance or behavior of the individual

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Genotype

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form of the gene responsible for the phenotypical characteristics

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mutagen

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chemical/radiation responsible for damaging DNA

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TAG

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-- genetically engineered
-- used to purify/identify certain proteins
-- TAGs enable us to express something in a cell that already has something similar to the TAG

-- take the gene that has the protein of interest, insert DNA, and encoding peptide & an epitome tag, introduce it to the cell

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Light Microscopy

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uses light to view very small objects

-- visible light (wavelength = 0.4-0.7 micrometers (bacteria & mitochondria)

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What is Live Cell Imaging? The types?

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a way of viewing live cells.
- bright field microscopy
- phase contrast microscopy
- dark-field microscopy

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Fluorescence Microscopy

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Tagging the protein with fluorescent antibodies & viewing them in a microscope that utilizes visible light

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Confocal Fluorescence Microscopy

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an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.[1] It enables the reconstruction of three-dimensional structures from the obtained images

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Multi-photon microscopy

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a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed

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What can you do with fluorescence?

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-- FRET (fluorescence resonance energy transfer)
-- Photoactivation
-- FRAP (fluorescence recovery after photobleaching)
-- Ion-sensitive Fluorescence indicators
-- Confocal Fluorescence Microscopy
-- Multi-photon microscopy
-- Tagging proteins for fluorescence microscopy
-- FACS (fluorescence activated cell sorter)

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Photoactivation

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Fluorescing a certain region and waiting to see how long it takes to diffuse away

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FRAP (Fluorescence Recovery After Photobleaching)

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-- opposite of photoactivation

-- photobleaching the spot and waiting to see how long it takes for the fluorescence to come back

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Ion-Sensitive Fluorescence Indicators

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Fluorescence Ca2+ indicators fluoresce at different wavelengths depending on whether they are bound to Ca2+ or not.

-- Helps to see where certain ions are found & can do a test for them after

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AFM (Atomic Force Microscopy)

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macromolecular microscopy - can look at DNA with it
-- there is a springy cantilever arm with an AMF tip at one end. this tip passes over the sample.
-- as it passes over, it goes up and down and this movement is detected by a change in the angle of the laser

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Electron Microscopy

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** must stain the sample with something that is electron-dense
-- 0.1 nm resolution; a much finer resolution
-- has magnetic coils for focusing
-- electrons scatter as they pass through material
-- lost electrons = dark area