Cell Biology Exam 1 (Topics 1-2)
Common Mass Spectrometry Methods
(ESI) electrospray ionization and (MALDI-TOF) matrix-assisted laser desorption ionization-time-of-flight
Problems of RNAi
-does not efficiently inactivate all genes -certain tissues can be resistant to RNAi -RNAi sometimes produces off-target effects by inactivating related genes
Two-Dimensional Gel Electrophoresis Step 1:
Sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, beta-mercaptoethanol and the denaturing agent urea (solution solubilizes, denatures and dissociates all the polypeptide chains but leaves their intrinsic charge)
Fluorescence Resonance Energy Transfer (FRET)
Technique for monitoring the closeness of two fluorescently labeled molecules (and thus their interaction) in cells, the energy transfer from the labeled proteins by illuminating the first protein and measuring the emission from the second
Secondary Culture
a cell culture where the cells are derived from a previous culture, as with immortalized cell lines
molecular cloning
isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated into a living organism
Light and Electron Microscopy Mutual Benefits
light microscopy can be used to very accurately locate specific molecules within a cell and the electron microscopy then allows the tagged molecule to be examined in its cellular context
Very High Speed Centrifugation
pellet contains ribosomes, viruses, large macromolecules
Low Speed Centrifugation
pellet contains whole cells, nuclei, and cytoskeletons
Gel-Filtration Chromatography
separates proteins based on size (porous beads let the bigger molecules through faster, while the smaller molecules get stuck in the pores and filter out last)
Two-Dimensional Gel Electrophoresis Isoelectric Focusing
separating polypeptide chains in a pH gradient which takes advantage of the variation in the net charge of a protein with the pH of the surrounding solution because the proteins separate to their characteristic isoelectric point where the protein has no net charge
fluorescence microscopy
uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation
Applications of of CRISPR/Cas9
1. Gene editing 2. Gene regulation 3. Base editing 4. RNA targeting 5. Chromatin topology 6. Chromatin imaging
Transmission Electron Microscope (TEM)
A microscope that uses an electron beam to study the internal structure of thinly sectioned specimens.
mass spectometry (MS)
- used as a detector to definitively identify solutes - measures mass to charge ratio - solutes show fragment pattern, which is compared to the published reference spectra -composed of the ion source, mass analyzer and a detector
CRISPR/Cas9 advantages
-relatively easy to design guide RNA (follows standard base pairing conventions -the gene to be controlled does not have to be modified (exploits DNA sequences already present in the genome -numerous genes can be controlled simultaneously (Cas9 expressed once with many guide RNA expressed in the same cell)
Affinity Chromatography
-uses specific interactions to slow down select molecules -can make use of receptor-ligand, enzyme-substrate, and antigen-antibody interactions to separate proteins (can create a nearly pure form from such specific interactions)
Transgenic Organisms
animals and plants that have been genetically engineered by gene deletion or gene replacement
Complementation Test DNA
used to ascertain whether a mutation falls in the same gene or in different genes, take a homozygous individual for the mutant gene in question and mates it with a homozygous individual for the other mutation (if they are on the same gene, all offspring will show the mutation but if not, the resulting offspring could show a normal phenotype because they retain a normal copy meaning they complement each other and restore a normal phenotype)
when is fluorescence microscopy most often used?
used to detect specific proteins or other molecules in the cells and tissues (FISH) -> reveals the cellular distribution and abundance of specific expressed RNA molecules in sectioned materials or in whole amounts of small organisms
electron microscopy
uses electrons instead of light, the shorter wavelength of electrons gives greater resolution, shooting electrons with shorter wavelengths to achieve a higher resolution in the picture
Complex Animal RNAi introduction
uses recombinant DNA techniques to make transgenic animals that express the RNAi under the control of an inducible promoter
Northern Blot
uses specific single stranded RNA probe to detect RNA expression level in the cell
Southern Blot
using specific single stranded DNA to detect DNA expression level in the cell
Restriction Nuclease Target Sequences
usually 4-8 nucleotide pairs which increases specificity of gene sites
Culture Shock
when culture conditions cause excessive stimulation from cell proliferation and the cell activates a poorly understood protective mechanism which stops cell division
Primary Culture
Cells are cultured directly from the tissues of an animal and have a limited life span.
General Steps of Protein Purification
1. Collect Intestinal Tissue from Experimental model 2. Mince Tissue and digest it into protein mix 3. Mix protein homogenate with protein assay to test if protein of interest is in the homogenate 4. Test whether the assay succeeded 5. Chromatography columns-> assay -> columns -> repeat
CRISPR/Cas9 Critical Requirements
1. Guide RNA provides the gene sequence specificity 2. Cas9 functions as the restriction enzyme to cut the DNA 3. Donor DNA that the experimenter designed provides the template for DNA repair
PCR General Steps
1. Heat to separate strands 2. Cool to anneal primers 3. DNA Synthesis (PCR is the method of choice for cloning relatively short DNA fragments)
By putting new DNA into animals we can...
1. Over express a gene 2. Knockout a gene
Hybridoma Cell Line Production Process
1. Propagate a clone of cells from a single antibody-secreting B lymphocyte to obtain a homogenous preparation of antibodies in large quantities 2. Fuse B-lymphocyte cells from immunized organism to the transformed B-lymphocyte 3. Propagate the hybridoma cells to create the permanent and stable source of a single type of monoclonal antibody
Equilibrium Binding Experiments
2 proteins are mixed at a range of concentrations, allowed to reach equilibrium, and the amount of bound complex is measured (half of the protein complex will be bound at a concentration that is equal to Kd), these experiments often involve the use of radioactive or fluorescent tags as one of the protein partners
restriction endonuclease
A bacterial enzyme that recognizes a specific DNA nucleotide sequence and that cuts the double helix at a specific site within the sequence.
Velocity Sedimentation
A method of separating subcellular components in a dilute salt solution. The tube is centrifuged, and the components sediment throughout the tube based upon size and shape when layered over a solution containing sucrose gradients (the bands must be protected from convective mixing to succeed)
Scanning Electron Microscope (SEM)
A microscope that uses an electron beam to scan the surface of a sample, coated with metal atoms, to study details of its topography, gives insight into its outer 3D shape
Polymerase Chain Reaction (PCR)
A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides (polymerase enzyme adds nucleotides to the 3' end of a growing strand of DNA but it requires a DNA primer which is a short nucleotide sequence that provides the 3' end from which synthesis can begin) PCR utilizes hybridization usually to synthesize the primer -at the start of each PCR cycle, the two strands of double stranded DNA are separated and a different primer is annealed to each which mark the right and left boundaries of the DNA to be amplified
SDS-PAGE
An electric field applied to a solution containing a protein molecule causes the protein to migrate at a rate that depends on its net charge and on its size and shape (uses a polyacrylamide gel as the inert matrix where proteins migrate and the gel can be prepared with specific pore sizes and the proteins are dissolved in sodium dodecyl sulfate, which is a powerful negatively charged detergent which causes proteins to unfold into extended polypeptide chains, individual protein molecules are released from associations to be analyzed separately)
Immortalized Cell Line
Cell line capable of an unlimited number of cell divisions, human cells generally coaxed to infinite proliferation by providing a gene that encodes the catalytic subunit of telomerase
Equilibrium Sedimentation
Centrifugal technique using a sucrose gradient to separate cellular components based on their buoyant density regardless of size and shape with the gradient and centrifugation separating the dense components (components of highest buoyant density are located at the bottom of the tube) - so selective that it can even separate macromolecules that contain different isotopes
What method is most often used to fractinate proteins?
Chromatography
Column Chromatography
Chromatography in which the substances to be separated are introduced onto the top of a column containing a matrix to separate the mixture based on various characteristics
CRISPR Acronym
Clustered Regularly Interspaced Short Palindromic Repeat
epistasis analysis
Comparing the phenotypes of different combinations of mutations to determine the order in which the genes act.
Flow of genetic material general
DNA -> Transcription (mRNA) -> Translation/Protein Synthesis (tRNA) -> proteins
Recombinant DNA
DNA produced by combining DNA from different sources
DNA polymerase
Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule, involved in the synthesis and repair of DNA strands, often used to create exact copies of existing DNA molecules in a lab setting and can even regenerate modified DNA with things like fluorescent or radioactive tags
Protein Purification Step 1:
Extracting a protein from the cells (cells can be broken up in various ways: osmotic shock, ultrasonic vibration, forced through a small hole, ground up in a blender etc) this breaks up many membranes into small fragments that immediately reseal into vesicles but if done correctly, leaves the organelles largely intact
GFP fusion protein
GFP gene introduction behind an endogenous gene --> fusion protein --> permits visualization of cellular gene products in vivo(WITHOUT antibody or fixation) viewed by fluorescence microscopy (CHROMOPHORE FORMED POST-TRANSLATIONALLY FROM PROTRUDING SIDE CHAINS OF 2 AA RESIDUES IN A SERIES OF AUTOCATALYTIC STEPS)
Replicative cell senescence
Phenomenon observed in primary cell cultures as they age, in which cell proliferation slows down and finally halts (reflects the progressive shortening and uncapping of the cell's telomeres)
bright field microscopy
Shines light through sample and magnifies the image with a series of lenses.
Epitope
Small, accessible portion of an antigen that can be recognized, antigenic determinant on a modified gene for tagging
Western Blot
The most common confirmation test for detecting protein expression level using antibodies after an SDS-PAGE by transferring the proteins to an sheet of nitrocellulose paper or membrane then soaking it in the tagged/fluorescent antibody to reveal the protein of interest
Dideoxy Sequencing
This method uses DNA polymerase, along with special chain-terminating nucleotides called dideoxyribonucleoside triphosphates to produce a collection of different DNA copies that terminate at every position in the original DNA sequence to determining the nucleotide sequence of a DNA fragment
How are a majority of proteins studied?
Tissues are not generally the preferred source of cells, more generally we use specific cell types grown in a culture (provide a more homogenous population of cells from which to extract materials)
Immunoprecipitation (IP)
To isolate proteins from complex mixtures and detect protein-protein interactions. Create a cell extract--add antibody for protein of interest--add large, insoluble beads that bind to the antibodies--centrifuge and remove soluble extract--elute protein of interest off of the insoluble beads. *Co-IP: Add antibodies for protein A and then immunoblot for protein B. Will only be able to see B if they the proteins interact.
Tandem mass spectrometry (MS/MS)
Using two mass spectrometers in tandem to determine the amino acid sequence of a single protein contained within a mixture of proteins, typically uses an electrospray ion source and then a quadrupole or ion trap to filter the other peptide identities out and identify the single peptide in a mix of many (also useful in detecting a precisely mapping post-translational modifications of proteins such as phosphorylation or acetylation)
CRISPR/Cas9
a bacterial system that can be used either to produce a mutation in a specific gene or to correct a mutation that is already present, uses a guide RNA sequence to target specific double-stranded DNA which it then cleaves and the gene coding key for this experiment (bacterial Cas9 protein) because it provides the environment to cleave the DNA
CRISPR
a collection of RNA sequences that tells Cas9 exactly where to cut
EcoR I
a common restriction enzyme produced by E. Coli. Cleave between G and A in following sequence, C-T-T-A-A-G- (reading 3' to 5')
Two-Dimensional Gel Electrophoresis
a method that separates the proteins in a sample into individual spots, combines two different separation procedures
Homogenate
a mixture of all of the components of the cell, but no intact cells, the thick slurry formed in step 1 of protein purification
Green Fluorescent Protein (GFP)
a protein that fluoresces green and is widely used in genetic analysis, two proteins of interest are labeled with different fluorescent proteins such that the emission spectrum of one fluorescent protein overlaps the absorption spectrum of the second (this causes an energy transfer if the proteins come very close to each other the energy of the absorbed light is transferred from one protein to the other to cause fluorescence)
Bacterial Artificial Chromosome (BAC)
another type of vector; allows for easier replication/manipulation as the number of genes is reduced to a smaller size or increased to a very large number
Monoclonal Antibody
antibody produced in a laboratory to attack antigens and to destroy cells; each antibody recognizes only a single type of antigenic site
Optical Sectioning with Light Sheet
based on wide field fluorescence microscopy, except the excitation light source is only a thin sheet of light where the eyepiece only sees the plane of fluorescent labels in the light
Hybridization
breaking the hydrogen bonds connecting complementary strands of DNA, but keeping the strand intact to study by heating the DNA sequence to 90*C before reattaching them by lowering the temperature
How do we trigger homologous recombination
by damaging the DNA strand and causing a break
Proteolytic Enzymes
catalyze the hydrolysis of peptide bonds (digests proteins in the extracellular matrix with agents that bind the Ca2+ on which cell-cell adhesion depends
Transformed Cell Lines
cell lines that are obtained from cancer cells. These cells can grow without attaching to a surface and can reach much higher cell densities
What is a normal imperfection that interferes with matrices in chromatography?
cellulose (causes uneven flow of solvent through the column that limits the resolution of conventional column chromatography)
Hybridoma cell lines
created to make monoclonal antibodies (production of unlimited quantities of identical antibodies and increases the specificity and convenience of antibody-based methods)
Perturbation
disruption in the genome and observing the changes in the associated phenotype
What happens in the SDS-PAGE?
each protein molecule binds large numbers of the negatively charged detergent molecules which mask the proteins intrinsic charge and causes it to migrate toward the positive electrode separating generally based on size because similar sizes bind similar numbers of detergent, smaller proteins move faster and farther down the gel than larger proteins
DNA ligase
enzyme that chemically links DNA fragments together, usually requires the inout of energy from a molecule like ATP
Restriction Nucleases
enzymes (proteins) that can cut DNA at specific sequences defined by the local nucleotide sequence, enabling the mixing of DNAs from different sources cut with the same enzyme (also called "molecular scissors")
GFP structure
fluorochrome shielded within the interior of beta barrel proteins
Optical Sectioning laser scanning confocal
focuses the light on a single focal point to observe only one spot with the maximum amount of light (better for thicker specimen)
epistatic
genes that mask the expression of other genes
Differential Interference Contrast Microscopy
highlights edges where there is a steep change of refractive index
Cas9 protein
holds the guide RNA, helps it basepair to DNA and cuts the genomic DNA
phase contrast light microscopy
in which phase alterations of light transmitted through the specimen are translated into brightness changes
RNA Interference (RNAi)
introduction of double-stranded RNA into a cell to inhibit gene expression, the RNA is processed and hybridized with the target-gene RNA thereby reducing the expression
Co-immunoprecipitation (Co-IP)
is a popular technique to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein
Organoids
miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy (derived from pluripotent embryonic stem cells)
Ion-Exchange Chromatography
molecules separated based on net surface charge (beads can carry either a positive or negative charge)
High Speed Centrifugation
pellet contains microsomes, small vesicles
Medium Speed Centrifugation
pellet contains mitochondria, lysosomes, peroxisomes
homologous recombination
process that results in genetic exchange between homologous DNA from two different sources (mother and father) which creates variability in germ cells
Chromatin Immunoprecipitation (ChIP)
proteins are covalently cross-linked to DNA in living cells which are broken open, and the DNA is sheared into small fragments and then purified by antibodies directed against a given transcription of the cross-linked DNA -> then the DNA is sequenced and all the sites occupied by the transcription regulator in the cell sample can be mapped across the cell's genome (responsible for specifying the particular pattern of GENE EXPRESSION)
Hydrophobic Chromatography
separates proteins based on hydrophobicity (beads have hydrophobic side chains to retard proteins with exposed hydrophobic regions)
Plasmid Vectors
small DNA circles that are physically separated from chromosomal DNA and can replicate independently, used for DNA cloning as a self-replicating genetic element
reverse transcription - PCR
synthesis of DNA from an RNA template using DNA primers for the chosen mRNA, then heating and cooling occurs for PCR amplification
Cell Fractination
takes cells apart and separates the major organelles from one another, separate cell components sometimes by size and density in a centrifuge (the fractinations are impure but will be further purified)
Recombinant DNA technology
the ability to manipulate DNA with precision in a test tube or an organism
Two-Dimensional Gel Electrophoresis Step 2:
the narrow tube of gel w/the separated proteins is soaked in SDS and placed on an SDS-PAGE slab, and then the electrophoresis is carried out so that we have the pH gradient separation horizontally and the SDS size migration running vertically
DNA Cloning
the production of multiple copies of a specific DNA segment and the amplification of a gene sequence
How to turn on/off a gene w Cas9
the protein complex will bind to a transcription activator or repressor to either turn on or off the gene that it is modifying
dark field microscopy
the specimen is lit from the side and only the scattered light is seen
Why do we uses SDS?
to denature and linearize proteins which allows them to run according to size, and to break down the disulfide bond connecting proteins