Biology 232 Lab FInal- Purdue 2023

Primary culture

Primary culture

the first step of cell culture and refers to the cells that are isolated from the tissue and proliferated under the appropriate artificial conditions

Cell line

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

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

culture medium

essential nutrients, carbohydrates, vitamins, minerals, amino acids, growth factors, hormones

For yeast we used YPD

Physio-chemical environment

temperature, osmotic pressure, pH/buffers, light intensity

Culture conditions

cell suspension, monolayer culture, anchorage-dependent

Yeast

Great model organism

Lag phase

number of cells remains relatively constant, cells are actively metabolizing but not dividing

Exponential phase (log phase)

cells grow most rapidly

stationary phase

metabolism slows and the cells stop rapid cell division, high cell density

Cell culture techniques

Spectrophotometry, hemocytometer, and spot plating

spectro- indirect method
spot and hemo - direct method

Uv effects on cell growth

Goal: measure the ability of yeast to grow under stress conditions with or without DNA repair mechanism.
Causes DNA damage
DNA repair: photoreactivation mechanism where PHR1 gene codes for photolyase is mutated in UV-S yeast

Types of Chromotography

Ion-exchange: solvent flows through positively charged beads and negatively charged molecules attach

Gel-filtration/gel permeation/ size exclusion: solvent flows through porous beads and smaller molecules get trapped in the beads and are thus slower than the larger molecules

Affinity chromotography: Solvent flows through beads with covalently attached substrates and enzyme molecules will bind (binding to little sticks on their surface)

SEC Advantages and disadvantages

Advantages - mild physiological conditions, simple, easily scalable - wide range of separation (many macromolecules)

Disadvantages - need 10% difference in size - limited by sample volume

Exclusion limit

the pore diameter defines the exclusion limit; proteins larger than the exclusion limit elute together in a single peak

void volume

large molecules are eluted in this

total volume

small molecules are eluted in this

elution volume

Volume of buffer needed to move a compound through the column

solutes within the separation range of the matrix are fractionally excluded

mobile phase of chromatography

where the molecules can move, always a liquid or gas

solvent used to move your components through

stationary phase of chromatography

solid, chromatography paper(in our case resin beads)

the solid part through which the components move through

Protein extraction steps

1) LYSIS: rupture of the cell membrane (TRIS of Phosphate buffer)
2) Centrifuge: spinning to separate cellular components
3) Dilution: dilute the sample
4)Analyze (graph)

Protein extraction reagents

Thiol compounds
Chelating agents
cations
substrates
protease inhibitors
osmotically active solutes
detergents
polyvinylpolyrr oidane

Thiol compounds

protects proteins from oxidation
examples: DTT

Protease inhibitors

suppression of endogenous proteases
Example: PMSF

chelating agents

molecules that attract or bind with other molecules and are therefore useful in either preventing or promoting movement of substances from place to place

Inhibits protease and eliminates contaminates
Example: EDTA

Osmotically active solutes

maintains tonicity of the solution comparable to that of the cell or tissue. avoids swelling or plasmolyzing of plastids and mitochondria
Example: Glycerol

Cations

maintains ionic strength or stabilizes

detergents

lysis
Example: Triton X-100, SDS

substrates

stabilizes enzymes

Polyvinylpolypyrrolidone (PVPP)

Plastic milled into fine particles
Removes phenolics from white wines that may have oxidized

Prevents "browning"
example: PVPP

Protein quantification methods

goal: measure the amount of proteins in a protein mix
Calorimetric dye-based assay
fluorescent dye methods

calorimetric dye-based assay

Fast and simple
lass compatible with detergent buffers
Uses the color change in a reaction to both
QUALITATIVELY (color change or not) and
QUANTITATIVELY (amount of color change) to draw conclusions
EXAMPLE: BRADFORD ASSAY - Uses coomassie dye-binding

fluorescent dye methods

Measures the increase in fluorescence which indicates the protein quantification in the sample

More sensitive (less sample required to test)

More complex equipment than a calorimetric dye-based assay

protein quantification

standard curve

Protein separation via SDS-PAGE

goal separate a mix of proteins by size

SDS-PAGE

denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel

PAGE- Poly-Acrylamide Gel Electrophoresis
- matrix of acrylamide and bisacrylamide
- higher bis concentration, smaller pores and better resolution
- cross-linking between bis and acrylamide needs a catalyst -commonly ammonium persulfate (generates the free radicals) + TEMED (stabilizes ions for polymerization)

Discontinuous gels

Stacking gel- lower acrylamide, allows for all protein samples to enter the separating gel at the same time (goes on top)

Separating gel- higher acrylamide, separates the proteins by size

Different gel dyes

Coomassie Blue- visual, quick, red/brown to blue

Silver staining- silver to brown/black, visual

Fluorescent dyes- UV/blue or green light makes it visible

ELISA

enzyme-linked immunosorbent assay
Goal: measure the levels of antigen in a solution

ELISA types

direct, indirect, sandwich

ELISA protocol

Coating: antigen absorbed onto well in ELISA plate in coating buffer

Blocking: A buffer containing unrelated proteins is used to block free sites

Detection: enzyme conjugates detection antibody binds antigen

Readout: substrate is catalyzed by enzyme to generate colored readout

Antibody components

There are two commonly used Coomassie dyes: Coomassie G-250 and Coomassie R-250 dyes. Which one is used in Bradford Assay and which one is preferred for staining protein gels?

Coomassie G250 is used in the Bradford Assay and R250 is preferred for staining protein gels.

Coomassie G-250 can exist in three forms: cationic (protonated), neutral and anionic (unprotonated). Which form of the dye is present in the Bradford Protein Assay Reagent? Which form is the dye in when bound to the protein?

In the Bradford Assay Reagent the dye is in its cationic form.(Red) When it is bound to the protein it is in its anionic form.

What problem are you likely to run into if the concentration of your Unknown protein is higher than the concentration of the standard protein dilutions used for building the standard curve? What solution to this problem can you offer?

The amount of protein in the sample will not be accurately estimated due to the protein concentration being higher than the standard curve. We can solve this problem using dilution to lead to a more accurate protein concentration.

What is the purpose of SDS in protein sample buffer and SDS-PAGE?

SDS denatures the native proteins by disrupting the hydrogen bonds. SDS-PAGE is a technique that allows proteins to be separated by their electrophoretic mobility.

What is the role of bis-acrylamide in polyacrylamide gel?

Bis-acrylamide is used to create crosslinks between the acrylamide to create polyacrylamide in gel electrophoresis.

What is the purpose of APS and TEMED in polyacrylamide gel casting?

APS and TEMED leads to the cross linking and polymerization of the acrylamide.