bio 232 final purdue

addition of thiol compounds to buffers

addition of thiol compounds to buffers

such as 2-mercaptoethanol - ADDED TO PROTECT PROTEINS FROM OXIDATION

chelating agents in buffers

protects enzymes from inactivation by heavy metal ions (such as EDTA)

addition of cations to buffers

help maintain ionic strength

addition of substrates to buffers

help stabilize enzymes

Protease inhibitors

helps suppress endogenous proteases

cell cycle checkpoints

G1 --> rest or divide
S --> is the DNA ok?
G2 --> Is the cell fully equipped?
M --> are sister chromatids fully separated and duplicated correctly?

what are cell cycle regulators

proteins that maintain and regulate cell cycle checkpoints

after a series of ten-fold dilutions, what will be the dilution of the final tube?

final dilution is 10^-7
dilution factor is 10^7

catabolism

breakdown of molecules to form simpler ones with the release of energy (oxidative process)

products of photosythesis and inputs of cellular respiration

sugars an oxygen

inputs of photosynthesis and products of cellular respiration

carbon dioxide and water

how can we calculate moles of CO2

liberation of CO2
consumption of O2

formula for calculation cell density

(total # of cells / # of squares) <b> dilution factor </b> volume factor = cells/mL

pigement

molecule that absorbs and reflects light

what are other pigments that we did not focus on in lab

carotenoids
anthocyanis
xantophylls

common solvents for chlorophyll and pigment extraction

acetone
ethanol
methanol
diethyl ether

Plasmid characteristics

small and circular DNA, extra chromosomal DNA, usually presents multiple copies in the cell, inherited and replicates

three main components for all plasmid vectors

1) origin of replication
2) heat shock/chemical transformation
3)recovery

competency

ability of bacteria to take up foreign DNA into cells

steps to DNA extraction

1) cell harvest + resuspension

2) cell lysis

3) neutralization buffer to remove cellular debris and separate plasmid DNA

4) DNA precipitation

5)DNA wash w ethanol to DE SALT DNA

6) rehydrate DNA

what foes the A260/A280 ratio signify - what is considered good

ratio of protein concentration

ratio for a pure extraction =
A260/A280 > 1.8

Bradford Assay

spectroscopic analytical procedure used to measure the concentration of protein in a solution

Protein extraction steps

1) Lysis
2) Centrifugation
3) Analysis
4) Dilution

two main components of any chromatography

mobile and stationary phase

what happens in mobile phase

This phase is made up of solvents•

It acts as a solvent-sample mixture that can be introduced in the column.•

It acts as a developing agent - helps in the separation of components in the sample to form bands.

• It acts as an eluting agent - the components that are separated during the experiment are removed from the column

• Some examples of mobile phase- are ethanol, acetone, and water.

what is the stationary phase

solid material w good absorption

* allows free flow of the mobile phase

in SEC what does the buffer do for the column?

allows mixture to travel through the column

how does SEC (size exclusion chromotagrpahy work)

* large molecules are luted first in the void volume

* small molecules are eluted in the total volume

* solutes within the separation range of the matrix are fractionally excluded

why is Triton - X used for protein isolation

helps soluble the membrane

extraction buffer components

thiol compounds
chelating agents
cations
substrates
protease inhibitors

thiol compounds

2-mercaptoethanol
- are frequently added to proteins to protect them from oxidation

Chelating agents

EDTA - protects enzymes from inactivation by heavy metal ions

cations

frequently added to maintain ionic strength (Mg+2)

substrates

added to stabilize enzymes and are therefore quite specific

protease inhibitors

supression of endogenous proteases

Osmotically active solutes

solutes that maintain tonicity of the solution comparable to that of the cell or tissue - acids swelling (SUCH AS GLYCEROL)

Detergents

often added to soluble organelles or membrane associated proteins

what macromolecules can ELISA quantify

antigens, antibodies, proteins and glycoproteins