Analyzing Cell, Molecules and Systems 1 Flashcards
What is the purpose of cell culture?
Study of cells, tissues, or organs in vitro. Also mimics the behaviour of the environment from where the cells were isolated
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
Where are primary cell cultures derived from?
From an explant, directly from the animal
Cultured either as tissue explants or single cells;
Can be embryo or adult; normal or neoplastic
How long does the primary cell culture usually survive?
Only for a finite period of time
What does primary cell culture involve?
Enzymatic and/or mechanical disruption of the tissue and some selection steps to isolate the cells of interest from a heterogeneous population
What is the main difference between primary and cell line cultures?
Primary: transient
Cell lines: permanent
What are continuous cell lines?
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 examples of Continuous cell lines?
CHO, SH-SY-5Y, Hela, K562, HEK293
How many times does a primary cell culture divide?
Divides only a limited number of times before losing their ability to proliferate
What is senescense?
Cells divide only a limited number of times before losing the ability to proliferate
Cell lines that have senescence are known as what?
Finite
What do primary cell cultures start out as?
A heterogeneous population
Cell line cultures are derived from what?
A primary or secondary culture
How are cell line cultures immortalized?
Spontaneous genetic mutation or by transformation vectors (viruses and/or plasmids)
What type of population are cell line cultures?
homogeneous
Cell line cultures will have ______ phenotype
Differentiated
What is the life span of cell line cultures?
Infinite life span in vitro, easy to grow and to cryopreserve for future experiments
Immortalied cell lines are isolated from naturally occurring cancer, major examples include:
human HeLa cells and mouse murine leukaemia cells
Mammalian cells in culture can be divided into 3 basic categories based on their morphology: describe
- Fibroblastic cells: bipolar or multipolar, have elongated shapes and grow attached to a substrate
- Epithelial-like cells:; polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches
- Lymphoblast-like cells: are spherical in shape and usually grown in suspension without attaching to a surface
What are the advantages of cell cultures?
- study of cell behaviour without the variations that occur in animals
- 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
What are the disadvantages of cell cultures?
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
What are the applications of cell cultures?
Basic research on cell/ gene function
Production of biological products (hormones, proteins, antibodies)
Testing of drugs, vaccines, chemical toxicity
Chromosomal or genetic analysis - clinical diagnostics
Regenerative medicine
What is hybridoma technology used for?
The generation of immortalized antibody-producing B cell lines, where an antibody-producing B cell is fused with a myeloma B cell cancer cell.
What is purification crucial for?
To study the structure and function of individual proteins
What technology is used to overexpress a protein?
Recombinant DNA technology
What type of proteins are purified?
endogenous
To reduce the complexity of the material, what usually is needed to be done?
Sub-cellular fractionation
Describe the steps of sub-cellular fractionation
Take the organ/tissue and mechanically blend
Homogenate: suspend different cell types
Centrifugation: separate different cell types by size and density
Lysis of cells: osmotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice
Ultracentrifugation: separate organelles
Now that organelle has been derived, how do you purify target protein from the organelle?
Column Chromatography
What are the three types of column chromatography?
Ion-exchange: based on charge
Gel-filtration: based on size
Affinity: based on ligand
Using affinity chromatography, how are PMCA isolated?
Using calmodulin sepharose beads
What is Tag?
An antigenic determinant or epitope which can be recognized by an antibody
Tag is used to purify the protein
How can SDS-PAGE be used to analyze individual proteins?
SDS is largely hydrophobic with a single negative charge. Its hydrophobic chain will get in crevices and unfold the protein.
It gives the protein a uniform charge: allows for all proteins to migrate towards a positive charge in the presence of a current
Once protein is denatured, it is run on a gel called SDS-PAGE gel.
What is also used in SDS process to further denature protein?
Beta-mercaptoethanol
What is the major method is used to identify unknown proteins?
Mass spectrometry
If you want to identify one protein among thousands, an you have no idea what you are looking for.
What is used in Mass Spectrometry to identify unknown proteins?
Chew up protein into protein fragments with tryptic digestion. Need to know the sizes of all the proteins our body makes that is sotred in computer databases. Analyze the size of tryptic fragments, and compare it to known sizes of proteins.
What can be used to identify post-translational modifications on proteins that are known?
Mass spectrometry
What method do you use to analyze specific proteins?
Western blotting
Describe western blotting method
Take the proteins on PAGE gel and transfer all the proteins out of gel to a membrane that sticks to the protein using electrophoretics
Once on membrane, primary and secondary antibodies are used to detect individual proteins
Describe Primary and secondary antibodies
Primary is used to recognize protein of interest. Secondary binds to the primary and conjugate it to some kind of marker so that we can detect the presence of that protein on the membrane
What method is used to analyze interacting proteins?
Immunoprecipitation: Utilize primary antibodies to identify protein complexes, or novel proteins bound to your protein of interest. Take protein Y and add an antibody to it and to find out what other proteins are bound you take immunoprecipitation.
Complexes of proteins can be pulled out using the antibody conjugated to some sort of heavy object such as magnetic beads. Take a magnet to the tube and pull out complex
How do you detect the presence of antibody that may be present due to infection, virus etc?
Indirect ELISA
What method is used to test HIV infection?
indirect ELISA
What method allows both the detection and the quantitation of antigen?
Sandwich ELISA
What ELISA method uses only a primary antibody conjugate?
Direct ELISA
