BIO 232 Final Exam (labs 7-12)

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