Cell culture
the process by which cells are grown under artificially controlled conditions, generally outside their natural environment
Primary Culture
The first step of cell culture refers to the cells being isolated from the tissue and proliferated under the appropriate artificial conditions
Utilize all the available nutrients and reach confluence
Cell line
After the first subculture, the primary culture becomes known as a cell line or subclone
Genotypic and phenotypic uniformity in the population
Cell strain
If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain
A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line
Culture medium
Essential nutrients, carbohydrates, vitamins, minerals, amino acids
Growth factors, hormones
Gasses (CO2, O2)
Physio-chemical environment
Temperature
Osmotic pressure
pH/Buffers
Light intensity
Culture conditions
Cell suspension
Monolayer culture
anchorage- dependent
Basal media
Contains amino acids, vitamins, inorganic salts, and a carbon source such as glucose
Must be further supplemented with serum
Serum media
Growth and adhesion factors, hormones, lipids, and minerals
regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements int the cell
Serum free media
Circumvents issues with using animal sera by replacing the serum with appropriate nutritional and hormonal formulations
Murashige and skoog medium (MS-media)
Basic media composition for plant cell cultures
Contains nutrients, carbon source such as sucrose
Lag phase
Number of cells remains relatively constant
cells are actively metabolizing but not dividing
Exponential phase
Cells grow most rapidly
Generation time
Stationary phase
Metabolism slows and the cells stop rapid cell division
High cell density
Spot plate method calculations
4 colonies in 10^-4 spot (5microL)
4×10^4 cells in original culture (5microL)
4× 10^4 × 200 cells in original culture (1mL)
= 8× 10^6 cells/ mL
Which type of radiation is absorbed by the ozone layer of Earth
UV-C
How does UV light damage genetic material?
UV-A and UV-B cause mutations in skin cells because the high energy and frequency an penetrate skin
Dimerization
Commonly caused by UV-B
Causes photochemical products in DNA
interferes with base pairing during DNA replication
Mutatuios through oxidative stress
creates free radical that then interacts with and oxidizes DNA bases
Oxidized bases don’t pair correctly during replication, leading to mutations
four types of DNA repair systems in yeast
Photoreactivation
Excision repair
Error-prone repair
Recombination repair
Excision repair
The damaged stretch of DNA is physically cut out and replaced with new DNA synthesis
Chromatography
A powerful analytical technique that separates mixtures into individual components
Two main types of chromatography
Liquid chromatography
gas chromatography
Thin-layer chromatography
Silica gel that is immobilized on a glass slide
Paper chromatography
Stationary phase is a thick filter paper and water drops settled in its pores, mobile phase is the appropriate fluid
Column chromatography
Stationary phase is a column on which the sample to be separated is loaded and then washed with mobile phase
Ion exchange chromatography
Uses electrostatic interactions between charged protein groups, and solid support material (matrix)
Matrix has an ion load opposite to that of the protein to be separated
Positively charged ion- exchange matrix - anion-exchange
Negatively charged matrix - cation-exchange
Affinity chromatography
The specific protein which makes a complex with the ligand attached to a solid support (matrix), and retained in the column, while free proteins leave the column. Usually an antigen-antibody interaction
The bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution
Gel permeation/Gel filtration/Size exclusion
Uses matric to seperate macromolecules based on their differences in molecular sizes
Matric is made from dextran, agrose, or polyacrylamide
Stationary phase is a column and consists of inert molecyles with small pores
How does Size exclusion work?
separation of molecules of different sizes
Large molecules do not enter the pores and are eluted first
Small molecules elute later
Factors that affect separation in size exclusion:
Flow rate
A high flow rate results in incomplete separation with simultaneous elution of proteins
Sample volume
Column length
Particle pore size
Protein Functions
Enzymes
Thousands of them, Rubsico, Alcohol dehydrogenase
Protein Functions
Structural proteins
Provides mechanical support
e.g. Collagen
Protein Functions
Motor proteins
Generate movement, myosin, flagellin
Protein Functions
Receptor proteins
Rhodopsin in retina, insulin receptor
Protein Functions
Storgage
Casein, endosperm
Protein Functions
Transport
Carry ions or small molecules
e.g. serum albumin
Protein Functions
Regulation
DNA binding proteins, transcriptional activators
Protein Functions
Signaling
Stress response hormones, growth factors. EGF
Protein Functions
Special purpose proteins
Selective advantage, highly variable
e.g. Antifreeze proteins, GFP
Protein Denaturation
A change in chemical structural and biological properties of proteins
What leads to protein denaturation?
Extreme temperatures (heat or radiation)
Changes in pH
Sonication or mechanical shearing
Chemicals; changes in salt concentration, solvents, alcohols, other chemical substances such as SDS
Effects of denaturation
Loss of stability
Unfolding
Permanent changes in the structure and folding of proteins
Loss of function
Most enzymes
Changes in binding sites
Substrate specifically
What is renaturation?
When a denatured protein returns to its original shape. Including regaining activity and globular proteins
What are qualities of a good buffer?
Low metal binding capabilities and relatively free of side effects
*What are common additions to extraction buffers?
Thiol compounds
Chelating agents
cations
substrates
protease inhibitors
osmotically active solutes
detergents
polyvinylpolypyrrolidone
*Thiol compounds:
Frequently added to protect proteins from oxidation
ex. DDT (dithiothreitol) or Beta-ME (2-mercaptoethanol)
*Chelating agents:
Protect enzymes from inactivation by heavy metals ions and prevents protein-metal ion aggregation/precipitation, substrate inhibition, or proteolysis
ex. EDTA (Ethylenediaminetetraacetic acid)
*Cations:
Frequently added to maintain ionic strength or provide specific stabilizing interactions
ex. K+, Na+, Mg2+
*Substrates:
Added to stabilize enzymes and therefore, quite specific
*Protease inhibitors:
Suppression of endogenous proteases
ex. PMSF (phenylmethylsulfonyl fluoride)
*Osmotically active solutes:
Maintains tonicity of the solution comparable to that of the cell or tissue. Avoids swelling or plasmolyzing of plastids and mitochondria
ex. Glycerol
*Detergents:
Often added to solubilize organelles or membrane-associated proteins
ex. Titron X-100
*Polyvinylpolypyrrolidone:
Generally added to plant extracts to prevent ‘browning’ from alkaloids and polyphenolic compounds
Describe Biuret assays:
Protein-copper chelation and secondary detection of reduced copper
Advantages:
Compatibility with most surfactants
linear response curve
Less protein-protein variation than the coomassie dye
Disadvantages:
Incompatible with substances that reduce copper
Incompatibility with common reducing agents
EX: BCA and Lowry assay
Describe calorimetric dye-based assays:
Protein dye binding and direct detection of the color change
fast and easy
Advantages:
Compatible with most salts, solvents, buffers, thiols, reducing substances, and metal-chelating agents
Disadvantages:
Incompatibility with surfactants
Protein-protein variation
EX: Bradford, (coomassie based)
Describe flourescent dye methods:
Protein-dye binding and direct detection of increase in fluorescence associated with the bound dye
Advantages:
Excellent sensitivity, requiring less protein sample for quantitation
Can be adapted for automated handling in high-throughput applications
Disadvantages:
Requires specialized instruments
Bradford Assay
Coomassie dye-binding assay
fast and simple protein quantification
Compatible with buffer salts, metal ions, reducing agents, chelators but less tolerant to detergents
Performed at room temperature and simple equipment is required
Detects protein concentration in the range of 2 to 1500 micrograms/mililiters
What is the reaction in a Bradford assay?
Proteins bind to the coomassie dye in an acidic environment, resulting in a color shift from reddish-brown to blue.
Electrophoresis
A powerful method for separating biomolecules in complex mixtures, based on the movement of charged particles in an electric field
Gel electrophoresis
A solid gel matrix is used for electrophoretic seperation of biological molecules
SDS-PAGE
A type of protein electrophoresis with protein migration through a gel under denaturing conditions
What are the dependent properties of particles when migrating in an electric field?
Size, shape, charge (positive or negative)
*SDS
separate proteins according to their electrophoretic mobility
*PAGE (Poly-Acrylamide Gel Electrophoresis)
A polyacrylamide gel consists of a matrix of acrylamide monomer cross-linked with bis-acrylamide to form a copolymer
Acrylamide forms long polyacrylamide chains that are cross-linked by the bis
The higher the concentration of bis, the more polyacrylamide polymers are cross-linked, and less gaps or pores
The higher the bis concentration, the smaller the pore size and hence the slower the proteins will migrate
The higher the percentage of gel, the better the resolution of smaller molecules
*Polyacrylamide gel
Linear polymer of acrylamide units cross-linked with bis-acrylamide to create a 3-dimensional matrix
The polymerization and cross-linking of acrylamide require a catalyst of free radical ions
Ammonium persulfate generates sulfate radical ions when dissolved
TEMED stabilizes these radical ions long enough for gel polymerization to occur
What is SDS-PAGE made of?
Majority of separating gel(higher acrylamide %, and higher pH), with the top being made of stacking gel (low acrylamide %, and lower pH)
What is the molecular sieving effect?
Small particles can move through the spaces in the mesh easier than large particles
Migration speed is inversely proportional to particle size
Gel pore size will affect the separation range of the proteins in your sample
Variations in % of acrylamide
Gel pore size depends on:
The percentage of acrylamide
Changing the ratio of acrylamide to bis-acrylamide also affects the separation range
A typical ratio of acrylamide to bis-acrylamide is 30:1
pH and ionic strength of the buffer system also affects the resolution and separation range
How to find the required field strength (E)
E=V/d
E = field strength
V = the voltage
d = the distance in cm between electrodes
Antigen:
defined as any molecule (usually a protein) that elicits an immune reaction
Antibodies:
Proteins that circulate in the blood and other body fluids, where they bind to specific antigens and mark them for destruction by phagocytic cells
Describe the antibody/immunoglobulin
Each antigen possesses features specific to its antibody
Epitopes or antigenic determinants: The actual portions or fragments of an antigen that react with receptors on B- B-lymphocytes and T-lymphocytes, as well as with free antibody molecules
Each protein or polysaccharide possesses many potential antigenic determinants
When a foreign protein is injected into an animal, antibodies to different determinants on the protein are produced and appear in the serum
This antibody-containing serum is called antiserum
*Each antibody consists of four polypeptides:
two heavy chains
Two light chains
Joined to form a y shaped molecule
*The amino acid sequence:
In the tips of the “y” varies greatly among different antibodies
*The variable region:
Composed of 110-130 amino acids, gives the antibody its specificity for binding antigen
Primary antibody
Antibody generated against the antigen
Secondary antibody
Antibodies that bind to the primary antibody
produced by injecting the primary antibody from one host species into a completely different host species
What is ELISA?
Enzyme Linked Immunosorbent Assay
Describe ELISA:
Antibodies are powerful tools to detect and quantify antigens in complex mixtures
All immunoassays are based on the specific binding of antibody to antigen
Engvall and Perlmann (1971), developed a method that enables the analysis of protein samples immobilized in microplate wells using specific antibodies
Applications of ELISA:
Can detect antigens, antibodies, proteins, and glycoproteins
Cost-effective
Increased sensitivity for diagnostic tests
Diagnosis of infection such as HIV, STDs, Bird flu, covid-19
Pregnancy tests,
What are some applications of ELISA?
Can detect antigens, antibodies, proteins, and glycoproteins
Cost-effective
Increased sensitivity for diagnostic tests
Diagnosis of infections such as HIV, STD’s, bird flu, COVID-19
Pregnancy tests
Measurement of cytokines or soluble receptors in cells
Concentrations of illicit drugs
food allergens
*How does ELISA work?
Binding antigens to the wells of a microtitration plate
Special plastics that bind proteins
Protein mixture containing the antigen is incubated in the wells so the antigen will be absorbed and immobilized
Binding of an enzyme-linked antibody to the immobilized antigen
An antibody to the antigen is incubated in the wells of the plate for the antibody-antigen interaction to take place
The antibody is covalently linked to an enzyme which serves as a marker molecule that makes it readily detectable
Commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphate (AP) or B-galactosidase
Detection of the antibody
A colorless substrate of the enzyme is added to the wells. The enzyme-linked to the bound antibody converts the colorless substrate to a colored product
*What are the types of ELISA?
Direct ELISA
Indirect ELISA
Sandwich ELISA
*Describe the basic steps of ELISA:
Coating
The antigen is absorbed onto well in ELISA plate in coating buffer
Blocking
A buffer containing unrelated protein is used to block free sites in the wells
Detection
Enzyme-conjugated detection antibody binds antigen
Readout
The substrate is catalyzed by the enzyme to generate a colored readout