Lecture 15: Analyzing Cell, Molecules and System 1 Flashcards
Cell culture refers to the
removal of cells from an organism, and promote their subsequent growth in a favorable artificial environment
Cell culture overview
- Revive frozen cell population isolate from tissue
- Maintain in culture (aseptic technique)
- Sub-culture (passaging)
- Cryopreservation
Primary cell cultures
- explant, directly form the animal
- usually only survive for a finite period of time
- involves enzymatic and/or mechanical disruption of the tissue and some selection steps to isolate the cells of interest from a heterogeneous population
Established or 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
- CHO (Chinese Hamster Ovary), SH-SY-5Y (human neuroblastoma derived), Hela (human cervical cancer), K562 (human erythroleukemia ), HEK293 (Human Embryonic Kidney)
most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called
replicative cell senescence
Divide only a limited number of times losing their ability to proliferate, which is a genetically determined event known as _____; these cell lines are known as finite
senescence
Immortalized Cell lines
- Introduction of a viral gene that partially changes the cell cycle regulation (e.g. the adenovirus E1 gene was used ot immortalize the HEK 293 cell line
- Artificial expression of key proteins required for immortality, for example telomerase which prevents degradation of chromosome ends during DNA replication in eukaryotes
Mammalian cells in culture can be divided into 3 basic categories based on their morphology:
- Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate
- Epithelial-like cells are 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 gorwn in suspension without attaching to a surface
What are the advantages of using cell cultures
- Study of cell behaviour without 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 cell cultures
- Have to develop standardised techniques in order to maintain healthy reproducible cells for experiments
- Takes time to learn aspetic 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 of the type of cells
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
Protein Purification is crucial to study the
- Structure and function fo individual proteins
It is challenging to isolate a single protein from thousands of others present in a cell but this is overcome using
- Recombinant DNA technology to overexpress a protein, thereby making purification easier
- Usually purification is started with sub-cellular fractionation in order to reduce the complexity of the material and then more specific purification techniques follow
Purification is normally started with
Sub-cellular fractionation, which reduces the complexity of the material and then more specific purification techniques follow
Cell can be broken up in various ways:
they can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or group up in a a blender. These procedures break many of the mbranes of the cell (including the PM and ER) into fragments that immediately reseal to form small closed vesicles. The suspension of cells is thereby reduced to a thick slurrey (called a homogenate or extract) that contains a varity of membrane enclosed organelles, each with a distincitve size, charge, and density
Preparative ultracentrifuge
- separates different components fo the homogenate by rotating at high speeds
- separates by size and density
- in genral, the largest objects experience the largest centrifugal force and move the most rapidly
after Low speed centrifugation (1000 times gravity for 10 minutes) the pellet (sediment contains)
whole cells, nuclei, and cytoskeletons
after medium-speed centrifugation (20,000 times gravity for 20 mintues) the pellet (sediment) of supernatant the supercontains
mitochondria, lysosomes, peroxisomes
After high-speed centrifugation (80,000 times gravity for 1 hour) the pellet (sediment) of the supernatant contains
microsomes, small vesicles
After ver-high speed centrifugation (150,000 times gravity for 3 hours) the pellet (sediment) of the supernatant contains
ribosomes, viruses, large, macromolecules
equilibrium sedimentation
- used to separate cell components on the basis of their buoyant density, independently of their size and shape.
- forms a series of bands, the bands closer to the bottom contain the components of highest buoyant density
- this method can be used to separate macromolecules that have incorporated heavy isotopes from the lighter isotopes
Sub-cellular fractionation
- Tissue: mechanical blending
- Homogenate: Suspension of different cell types
- Centrifugation to separate different cell types
- based on size and density
- Lysis of cells: osomotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice
- Ultracentrifugation: separate organelles
Lipid rafts
- PM microbdomains enriched in cholesterol, sphingolipids, and gangliosides
- Detergent insoluble, low buoyant density
- Local centers for signal transduction processes
- Sites for abnormal processing of proteins in neurodegenerative disorders
Types of column chromatography
- Ion-echange chromatography
- separated according to their charge
- Hydrophobic chromatography
- by hydrophobicity
- gel-filtration chromatography
- size
- affinity chromatography
- ability to bind to particular small molecules or to other macro-molecules
In gel-filtation chromatography what moves faster smaller or larger molecules
larger
affinity chromatography uses antibodies and then a _____ to break the bonds
reducing agent
protein purification by chromatography is done using
several different kinds of columns with affinity chromatography being the most efficient (each time you pool the fraction number that shows enzymatic ativity and use it in the next column)
using recombinant DNA technology any gene can be modified to produce a protein with a
- special recognition tag
- Tag is an antigenic determinant or epitope which can be reognized by an antibody
- Use tag to purify the protein
what are some examples of special regonition Tags used to purify the protein
- His tag (copper or nickel affinity chromatography)
- Enzyme tag (Glutathione S Transferase)
- Tandem affinity purification (TAP) tagging. Cleavage site built in the fusion protein
SDS is largely hydrophobic with _______ negative charge
a single negative charge
SDS-phage
- proteins migrate at a rate that depends on its net charge and on its size and shape
- it uses a highly cross-linked gel of polyacrylamide as the inert matrix thorough which the proteins migrate.
- The proteins are disolved in a solution that inculdes a powerful negatively charged detergent, sodium dodecyl sulfate, or SDS
- binds hydrophobic parts of proteins
- unfolds proteins
- reducing agent beta-mercaptoethanol is usually added to beak any S-S linkages in the proteins
- gives uniform charge
- allows for all proteins to migrate toward a positive charge in the presence of current
two-dimensional gel electrophoresis combines two different separation procedures, can resolve up to ____ protiens in the form of a two-dimensional protein map
2000
SDS-PAGE can detect resolved proteins with stains ____ being the most common
Coomassie bleu stain (not that bigger mass don’t travel as far on the gel)
Separation of protein molcules by isoelectric focusing
- First dimension of two-dimensional polyacrylamide-gel electrophoresis
- taks advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution
- at low pH( high H+ concentration) the carboxylic acid groups of proteins tend to be uncharged (-COOH) and their nitrogen containing basic groups fully charged, giving most proteins a net positive charge. At high pH, the carboxylic acid groups are negatively charged (-COO-) and the basic groups tend to be uncharged, giving most proteins a negative charge . At its isolectric pH, a protein has no net charge since the positive and negative charges balance out. Thus, when a tube containing a fixed pH gradient is subjected to a strong electric field in the appropriate direction, each protein species migrates until it forms a sharp band at its isolectric pH
In two dimensional polyacrylamide-gel electrophoresis the only proteins left unresolved
are those that have both identical sizes and identical isoelectrical points, a relatively rare situation
The two-dimensional polyacrylamide-gel electrophoresis has what two dimentsion
- First separated on the basis of their isoolectric points by isoelectric focusing in the orizontal dimension (basic side is left and acidic is to the right)
- They are further fractionated according to their molecular mass by electrophoresis from top to bottom
- bigger spot means more of that specific protein
_____ provides a highly sensitive method for identifying unkown proteins
Mass Spectrometry
Mass spectrometry requies
Tryptic digestion products (peptide fragments), ionization (charge) a detection method (time of flight) and computer databases with known protein sizes
Mass spectrometry is performed using complex instruments with three major components
- ion source
- transforms tiny amounts of peptide sample into a gas containing individual charged peptide molcules
- Mass analyzer
- The ions are acellerated by an electric field into the mass analyzer
- where electric or magnetic fields are used to separate the ions on the basis of their mass-to-change ratios
- Detector
- separated ions collide with a detector
- generates a mass spectrum containing a series of peaks representing the masses of the molecules in the sample
tandeme mass specrometry typically involves two mass analyzers separated by a collision chamber containing
an inert, high-energy gas. (used to cause peptide fragmentation)
________ is useful for detecting and precisely mapping post-translational modifications of proteins, such as phosphorylations or lysine acetylations of mitochondiral proteins
tandem mass spectrometry
Sirt3 is a mitochondrial specific protein ____
- deacetylase
- loss of expression results in an increase in acetylated Sirt3 substrates
A key method for identifying proteins that bind to one another tightly is ______
- Co-immunoprecipitation
- a specific targe portien is immunoprecipitated from a cell lysate using specific antibodies coupled to beads. If the target protein is associated tightly enough with another protein when it is captured by the antibody, the partner precipitates as well and can be idnetifed by mass spectrometry
- this method is ufesful for identifying proteins that are part of a complex inside cells, including those that interact only transiently- for example, when extracellular signal molecules stimulate cells
Western Blotting
- Technique by which proteins are separated by electrophoresis and immobilzed on a paper sheet and then analyzed usually by means fo a labeled antibody. Also called immunoblotting
- steps
- resolve protein samples on native PAGE
- Electrophoretically transfer fractionated proteins from gel onto PVDF membrane
- Block the mebrane with neutral protein (BSA or milk casein)
- incubate the membrane with HRP-labled antibody specific to prey protein
- incubate the blot with chemiluminescent HRP substrate and expose to film
what is indirect immuno-cytochemistry used in western blotting
- The primary antibody is itself recognized by many molcules of the secondary antibody. The secondary antibody 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)
Antibodies can be used to quantify the amount of an antigen in a smaple mixture by the technique called
Enzyme-linked immunosorbent assay (ELISA)
ELISA is carried out in multi-well plates in which samples are added, thus, samples are nto separated. If the antigen is present, the
- antibody-enzyme complex will bidn to it, and the enzyme component of the antibody-enzyme complex will catalyze the reaction generatign the colored product. Thus, the presence of the colored product indicates the presence of the antigen
- the color is measured by absorbance of a light source in a plate reader
What is the basis of the test for HIV infection
- indirect ELISA to detect the presence of antibody
- in that test, viral core proteins (the antigen) are absorbed to the bottom of a well. Antibodies form a patient are then added to the coated well and allowed to bind to the antigen
- Finally, enzyme-linked antibodies to human antibodies (for instance, goat anibodies that recognize human antibodies) 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 utnr impleis that the patient had antibodies to the viral antigen
What kind of ELISA is used for pregnancy test
Sandwich ELISA
Snadwich ELISA
- Allowing both the detection and the quantitation of antigen
- 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 antgen present
- Consequently, it permits the measurement of small quantities fo antigen
- To calculate the absolute amount of antigen, a standard curve with known amount of antigen is required
is ELISA used to detect troponin
yes
X-ray crystallography
- Patter in which x-ray waves are diffracted can tell us about shape and atomic distances within the molcule
- Limited by the ability to crystallize the protein
Nuclear Magnetic Resonance (NMR)
- Similar to an MRI you can get at a hospital or clinic
- Can analyze proteins in solution (more relevant)
- Resolving small proteins or domains requires high powered magnets (300-900MHz) limited by size
