BIO 232 Final Exam (labs 7-12)

the process by which cells are grown under artificially controlled conditions, generally outside their natural environment

• 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

• After the first subculture, the primary culture becomes known as a cell line or subclone

• Genotypic and phenotypic uniformity in the population

• 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

• Essential nutrients, carbohydrates, vitamins, minerals, amino acids

• Growth factors, hormones

• Gasses (CO2, O2)

• Temperature

• Osmotic pressure

• pH/Buffers

• Light intensity

• Cell suspension

• Monolayer culture

• anchorage- dependent

• Contains amino acids, vitamins, inorganic salts, and a carbon source such as glucose

  • Must be further supplemented with serum

• 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

• Circumvents issues with using animal sera by replacing the serum with appropriate nutritional and hormonal formulations

• Basic media composition for plant cell cultures

• Contains nutrients, carbon source such as sucrose

• Number of cells remains relatively constant

• cells are actively metabolizing but not dividing

• Cells grow most rapidly

• Generation time

• Metabolism slows and the cells stop rapid cell division

• High cell density

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

UV-C

UV-A and UV-B cause mutations in skin cells because the high energy and frequency an penetrate skin

Commonly caused by UV-B

Causes photochemical products in DNA

• interferes with base pairing during DNA replication

• creates free radical that then interacts with and oxidizes DNA bases

• Oxidized bases don’t pair correctly during replication, leading to mutations

Photoreactivation

Excision repair

Error-prone repair

Recombination repair

• The damaged stretch of DNA is physically cut out and replaced with new DNA synthesis

A powerful analytical technique that separates mixtures into individual components

Liquid chromatography

gas chromatography

Silica gel that is immobilized on a glass slide

Stationary phase is a thick filter paper and water drops settled in its pores, mobile phase is the appropriate fluid

Stationary phase is a column on which the sample to be separated is loaded and then washed with mobile phase

• 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

• 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

• 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

• separation of molecules of different sizes

  • Large molecules do not enter the pores and are eluted first

  • Small molecules elute later

• Flow rate

  • A high flow rate results in incomplete separation with simultaneous elution of proteins

• Sample volume

• Column length

• Particle pore size

Thousands of them, Rubsico, Alcohol dehydrogenase

Provides mechanical support

e.g. Collagen

Generate movement, myosin, flagellin

Rhodopsin in retina, insulin receptor

Casein, endosperm

Carry ions or small molecules

e.g. serum albumin

DNA binding proteins, transcriptional activators

Stress response hormones, growth factors. EGF

Selective advantage, highly variable

e.g. Antifreeze proteins, GFP

A change in chemical structural and biological properties of proteins

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

Loss of stability

• Unfolding

• Permanent changes in the structure and folding of proteins

Loss of function

• Most enzymes

• Changes in binding sites

• Substrate specifically

When a denatured protein returns to its original shape. Including regaining activity and globular proteins

Low metal binding capabilities and relatively free of side effects

Thiol compounds

Chelating agents

cations

substrates

protease inhibitors

osmotically active solutes

detergents

polyvinylpolypyrrolidone

Frequently added to protect proteins from oxidation

ex. DDT (dithiothreitol) or Beta-ME (2-mercaptoethanol)

Protect enzymes from inactivation by heavy metals ions and prevents protein-metal ion aggregation/precipitation, substrate inhibition, or proteolysis

ex. EDTA (Ethylenediaminetetraacetic acid)

Frequently added to maintain ionic strength or provide specific stabilizing interactions

ex. K+, Na+, Mg2+

Added to stabilize enzymes and therefore, quite specific

Suppression of endogenous proteases

ex. PMSF (phenylmethylsulfonyl fluoride)

Maintains tonicity of the solution comparable to that of the cell or tissue. Avoids swelling or plasmolyzing of plastids and mitochondria

ex. Glycerol

Often added to solubilize organelles or membrane-associated proteins

ex. Titron X-100

Generally added to plant extracts to prevent ‘browning’ from alkaloids and polyphenolic compounds

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

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)

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

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

Proteins bind to the coomassie dye in an acidic environment, resulting in a color shift from reddish-brown to blue.

A powerful method for separating biomolecules in complex mixtures, based on the movement of charged particles in an electric field

A solid gel matrix is used for electrophoretic seperation of biological molecules

A type of protein electrophoresis with protein migration through a gel under denaturing conditions

Size, shape, charge (positive or negative)

separate proteins according to their electrophoretic mobility

• 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

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

Majority of separating gel(higher acrylamide %, and higher pH), with the top being made of stacking gel (low acrylamide %, and lower pH)

• 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

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

E=V/d

E = field strength

V = the voltage

d = the distance in cm between electrodes

defined as any molecule (usually a protein) that elicits an immune reaction

Proteins that circulate in the blood and other body fluids, where they bind to specific antigens and mark them for destruction by phagocytic cells

• 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

• two heavy chains

• Two light chains

• Joined to form a y shaped molecule

In the tips of the “y” varies greatly among different antibodies

• Composed of 110-130 amino acids, gives the antibody its specificity for binding antigen

Antibody generated against the antigen

Antibodies that bind to the primary antibody

produced by injecting the primary antibody from one host species into a completely different host species

Enzyme Linked Immunosorbent Assay

• 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

• 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,

• 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

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

Direct ELISA

Indirect ELISA

Sandwich 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