Analyzing Cell, Molecules, and Systems 1 Flashcards
What happened in cell culture discoveries in 1948?
First use of antibiotics in tissue culture by Keilova.
What happened in cell culture discoveries in 1952?
Development of HeLa cell line by Gey, et al.
What happened in cell culture discoveries in 1964?
Discovery of pluripotency of embryonic stem cells by Kleinsmith and Pierce.
What happened in cell culture discoveries in 1990?
Application of cell culture to production of biotherapeutic agents.
What happened in cell culture discoveries in 2000?
Mapping of the human genome.
What happened in cell culture discoveries in 2008?
Era of induced pluripotent stem cells (iPSc’s) - promises and challenges.
What does cell culture refer to?
Refers to the removal of cells from an organism, and promote their subsequent growth in a favorable artificial environment.
What is a primary cell culture derived from?
Derived from an explant, directly from the animal.
How long does a primary cell culture survive?
Only survive for a finite period of time.
True or False:
Primary cell culture involves enzymatic and/or mechanical disruption of the tissue ad some selection steps to isolate the cells of interest from a heterogeneous population.
True
What is a estblished or continuous cell line?
A primary culture that has BECOME IMMORTAL due to some transformation.
> most commonly tumour derived, or transformed with a virus such as Epstein-Barr.
What are some common established or continuous cell lines that are commonly used in laboratories?
- CHO (Chinese Hamster Ovary)
- SH-SY-5Y (human neuroblastoma derived)
- He-La (human cervical cancer)
- K562 (human erythroleukemia)
- HEK293 (Human Embryonic Kidney)
What is a primary cell culture?
Divides only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as SENESCENCE; these cell lines are known as FINITE.
Where are cells that are used to start primary cell cultures derived from?
Directly from animal tissue - embryo or adult? Normal or neoplastic?
What are primary cell cultures cultured as?
Either as tissue explants or single cells; which start as a heterogeneous population.
Do cells that are used to start a primary cell culture started as a heterogeneous population?
Yes - start as a heterogeneous population.
What are cell line cultures derived from?
primary or secondary culture
Are cell line cultures mortal or immortalized?
immortalized - spontaneous genetic mutation or by transformation vectors
What causes immortalization of cell line cultures?
spontaneous genetic mutation or by transformation vectors (e.g., viruses and/or plasmids)
True or False:
Cell line cultures have a “more” differentiated phenotype.
True
True or False:
Cell line cultures have an infinite life span in vitro and are easy to grow and to cryopreserve for future experiments.
True
Are cell line cultures commercially available or obtained by collaborators?
Yes (American Type Culture Collection (ATCC) or other companies))
What cell line culture is commonly used as a Parkinson’s model in the laboratory?
SH-SY5Y - Neuroblastoma
What is a immortalized cell line?
- introduction of a viral gene that partially changes the cell cycle regulation (e.g., the adenovirus E1 gene was used to immortalize the HEK 293 cell line).
- artificial expression of key proteins required for immortality (e.g., telomerase which prevents degradation of chromosome ends during DNA replication in eukaryotes).
What are the 3 basic categories of mammalian cells in culture that are based on their morphology?
1) fibroblastic (or fibroblast-like) cells
2) epithelial-like cells
3) lymphoblast-like cells
What are fibroblastic (or fibroblast-like) cells?
> bipolar or multipolar
> have elongated shapes
> grow attached to a substrate
What are epithelial-like cells?
> polygonal in shape with more regular dimensions
> grow attached to a substrate in discrete patches
What are lymphoblast-like cells?
> spherical in shape
> usually grown in suspension without attaching to a surface
Which cell category are grown in suspension without attaching to a surface?
lymphoblast-like cells
Which cell category grows attached to a substrate in discrete patches?
epithelial-like cells
Which cell category grows attached to a substrate, but not in discrete patches?
fibroblastic (or fibroblast-like) cells
What are the advantages of a cell line culture?
- study of cell behaviour without the variations that occur in animal
- characteristics of cells can be maintained over several generations, leading to good reproducibility between experiments
- control of the growth environment leads to uniformity of sample
- cultures can be exposed to reagents (e.g., radio-chemicals or drugs at defined concentrations)
What are the disadvantages of a cell line culture?
- have to develop standardized techniques in order to maintain healthy reproducible cells for experiments.
- takes time to learn aseptic technique.
- quantity of material is limited.
- dedifferentiation and selection can occur and many of the original cellular mechanisms can be lost.
- it can be costly depending on the type of cells.
What are the applications of a cell line culture?
- basic research on cell/gene function
- production of biological products (hormones, proteins, antibodies).
- testing of drugs, vaccines, and chemical toxicity.
- chromosomal or genetic analysis - clinical diagnostics.
- regenerative medicine
Why is protein purification difficult?
Challenging to isolate a single protein from thousands of others present in a cell.
Why is protein purification important?
Purification is crucial to study the structure and function of individual proteins.
What technique is used to overexpress a protein so that it can be purified?
recombinant DNA technology
What type of proteins are purified?
endogenous proteins
To purify endogenous proteins, what type of technique usually needs to be performed?
sub-cellular fractionation
What does sub-cellular fractionation do to the material?
reduces the complexity of the material, which is then followed by purification
What are the two types of ultracentrifuge used in the laboratory?
- fixed angle rotor
- swinging bucket rotor
What is sub-cellular fractionation?
> mechanical blending of tissue
> homogenate: suspension of different cell types
> centrifugation to separate different cell types, based on size and density.
> lysis of cells: osmotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice.
> ultracentrifugation: separate organelles
ended on slide 19
cell fractionation by centrifugation
What is a PM microdomain that is enriched in cholesterol, sphingolipids, and gangliosides?
lipid raft
Are lipid rafts detergent insoluble?
Yes
What function do lipid rafts have?
Are local centers for signal transduction processes.
How are lipid rafts involved in neurodegenerative disorders?
sites for abnormal processing of proteins
True or False:
Lipid rafts are detergent insoluble with low buoyant density.
True
Walk through the isolation of synaptic plasma membrane (SPMs).
Brain -> homogenize -> centrifuge (3000 rpm x 5 min) -> keep pellet -> centrifuge pellet (10,000 rpm x 10 min) -> keep both pellet and supernatant:
> Pellet -> homogenize, density gradient centrifugation -> synaptosomes 8-14% Ficoll -> lysis -> synaptic PMs, density gradient centrifugation -> lipid rafts
> supernatant -> centrifuge (90,000 rpm x 60 min) -> microsomes
What makes up a synaptosome?
- presynaptic neuron
- synaptic cleft
- postsynaptic neuron
What is homogenate?
suspension of different cell types
What are the steps of cell fractionation by centrifugation?
cell homogenate -> low-speed centrifugation -> results in pellet and supernatant.
- pellet 1 -> contains whole cells nuclei cytoskeletons
- supernatant subjected to medium-speed centrifugation -> results in pellet 2 and supernatant.
> pellet 2 contains mitochondria, lysosomes, and peroxisomes
> supernatant subjected to high-speed centrifugation -> results in pellet 3 and supernatant.
- pellet 3 contains microsomes and small vesicles
- supernatant subjected to very high-speed centrifugation -> results in pellet 4
- pellet 4 contains ribosomes, viruses, and large macromolecules.
In cell fractionation by centrifugation, what does the first pellet contain?
whole cells nuclei cytoskeletons
In cell fractionation by centrifugation, what does the second pellet in the process contain?
mitochondria
lysosomes
peroxisomes
In cell fractionation by centrifugation, what does the third pellet in the process contain?
microsomes and small vesicles
In cell fractionation by centrifugation, what does the fourth pellet in the process contain?
ribosomes
viruses
large macromolecules
What are the 3 different matrices for column chromatography?
> ion-exchange chromatography
> gel-filtration chromatography
> affinity chromatography
In which type of column chromatoraphy can a antibody be used in?
affinity chromatography
Which type of chromatography is used in the purification of PMCA from SPMs?
affinity chromatography
How is recombinant DNA technology used in the process of genetically engineering proteins?
Using recombinant DNA technology any gene can be modified to produce a protein with a special recognition tag.
What is the tag that is placed on a gene by recombinant DNA technology?
Is an antigenic determinant or epitope which can be recognized by an antibody.
What are some examples of tags added to genetically engineered proteins?
> His tag (copper or nickel affinity chromatography)
> Enzyme tag (Glutathione S Transferase)
> Tandem affinity purification (TAP) tagging. (cleavage site built in the fusion protein)
What is SDS?
SDS is largely hydrophobic with a single negative charge.
What does SDS do?
> unfolds proteins
gives uniform charge
- allows for all proteins to migrate toward a positive charge in the presence of current
What is the function of beta-mercaptoethanol?
reduces disulfides (further denature protein)
What is a common stain used to detect resolved proteins in a SDS-page gel?
Coomassie blue
What is the separation of proteins via 2-D gel electrophoresis based on?
requires SDS, but is based on separating amino acids on their pI
At a low pH, will the protein be positively or negatively charged?
positively charged
At a high pH, will the protein be positively or negatively charged?
negatively charged
What happens to the protein when it reaches the pH of its isoelectric point?
the protein has no net charge and therefore no longer migrates in the electric field
What is mass spectrometry used for?
To identify unknown proteins.
can also identify post-translational modifications
What does mass spectrometry require?
Requires Tryptic digestion products (peptide fragments), ionization (charge) a detection method (time of flight) and computer databases with known protein sizes.
trypsin cleaves after hydrophobic amino acids
What are the two different types of mass spectrometry?
> standard mass spectrometry (MS)
> tandem mass spectrometry (MS/MS)
What is identified by mass spectrometry in the identification of post-translational modifications?
lysine acetylation of mitochondrial proteins
What is Sirt3?
A mitochondrial specific protein deacetylase.
What does the loss of Sirt3 expression cause?
Loss of expression results in an increase in acetylated Sirt3 substrates.
What technique is used to analyze specific proteins?
Western blotting
What are the 6 steps of Western blotting?
1) resolve protein samples on native PAGE
2) electrophoretically transfer fractionated proteins from gel onto PVDF membrane
3) block the membrane with neutral protein (BSA or milk casein)
4)
5) incubate the membrane with HRP-labeled antibody specific to prey protein
6) incubate the blot with chemiluminescent HRP substrate and expose to film
Analyzing specific proteins - Western blotting
Indirect immuno-cytochemistry: this detection method is very sensitive because the primary Ab is itself recognized by many molecules of the secondary Ab. The secondary Ab is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent dyes (for fluorescence microscopy), and the enzyme horseradish peroxidase (for either conventional light microscopy or electron microscopy and biomedical detection).
What technique is used to analyze interacting proteins?
Immunoprecipitation
- can isolate one protein from a mixture of many proteins
- can find “co-IP” partners to identify protein complexes
True or False:
Proteins can be detected and quantitated by ELISA.
True
What is enzyme-linked immunosorbent assay (ELISA) used for?
Antibodies can be used to quantify the amount of an antigen in a sample mixture by ELISA.
What is ELISA carried out in?
multi-well plates (samples are not separated)
If the antigen is present in the sample, what happens?
The Ab-enzyme complex will bind to it, and the enzyme component of the Ab-enzyme complex will catalyze the reaction generating the the colored product.
Thus, the presence of the colored produce indicates the presence of the Ag.
In an ELISA, what does the presence of a colored product indicate?
The presence of the antigen.
How is color measured in an ELISA?
Measured by absorbance of a light source in a plate reader.
True or False:
Such an enzyme-linked immunosorbent assay, which is rapid and convenient, can detect less than a nanogram (10-9 g) of a protein.
True
What is a indirect ELISA used for?
To detect the presence of antibody and is the basis of the test for HIV infection.
How does the indirect ELISA test work to test patients for HIV?
> In that test, viral core proteins (the antigen) are absorbed to the bottom of a well. Abs from a patient are then added to the coated well and allowed to bind to the Ag.
> finally, enzyme-linked Abs to human Abs (for instance, goat Abs that recognize human Abs) are allowed to react in the well and UNBOUND ANTIBODIES ARE REMOVED BY WASHING. Substrate is then applied.
> an enzyme reaction suggests that the enzyme-linked antibodies were bound to human antibodies, which in turn implies that the patient had antibodies to the viral antigen.
What does a sandwich ELISA allow?
Both the detection and the quantitation of antigen.
What are the steps of a sandwich ELISA?
> antibody to a particular antigen is first absorbed to the bottom of a well.
> next, the antigen (or blood or urine containing the antigen) is added to the well and binds to the antibody.
> finally, a second, different antibody to the antigen is added. This antibody is enzyme linked and is processed as described for indirect ELISA.
> in this case, the extent of reaction is directly proportional to the amount of antigen present.
> consequently, it permits the measurement of small quantities of antigen.
> to calculate the absolute amount of antigen, a standard curve with known amount of antigen is required.
Which ELISA is used for an HIV test?
Indirect ELISA
What common test used by women is a sandwich ELISA?
pregnancy test
1) free monoclonal antibody specific for hCG, a hormone produced during pregnancy.
2) capture monoclonal antibody bound to substrate.
3) sandwich formed by combination of capture antibody and free antibody when hCG is present, creating a color change.
What is X-ray crystallography limited by?
Limited by the ability to crystallize the protein.
What is X-ray crystallography used to do?
Analyze Protein Structure
How can X-ray crystallography tell us about protein structure?
The pattern in which x-ray waves are diffracted can tell us about shape and atomic distances within the molecule.
Can nuclear magnetic resonance (NMR) analyze proteins in solution?
Yes
What is NMR limited by?
size - resolving small proteins or domains requires high powered magnets (300-900 MHz)
