Cell Bio (BI220) lab practical- Butler university

8 steps to western blot

1. sample prep 2. gel electrophoresis 3. blotting 4. blocking 5. antibody probing 6. detection 7. imaging 8. analysis

what is the enzyme that digests lactose

B-galactosidase

4 components of HeLa medium

BSA penicillin glutamine pyruvate

indicator dye to monitor ETC

DCPIP

negative control

DMSO; doesn't effect experiment

fluorescence microscope

Instrument used to visualize a specimen that has been labeled with a fluorescent dye; samples are illuminated with a wavelength of light that excites the dye, causing it to fluoresce. beneficial to see only specific compounds

kingdoms

Plants, Animals, Protists, Fungi, Archaebacteria, Eubacteria

what is control measured in

RPM

why is gravity reported

RPM changes

why is absorbance used instead of %T

absorbance directly proportional to concentration

p value greater than 0.05

accept null hypothesis; is not significant

excitation

allow light through narrow band of wavelength; excites fluorescent molecule

coomassie blue stain

allow you to see bands representing most prominent proteins in gel

control experiment

an experiment designed to control for variables affecting the results of another experiment

antibody exposure

apply detector compound to see microtubules

why must tubes in centrifuge be opposite from eachother

balance; can ruin centrifuge otherwise fragments can fly and hit walls, humans, etc

secondary antibody

binds to primary antibody

DAPI

blue stain; helps see nucleus by binding to DNA and detect apoptosis

bromophenol blue

blue tracking dye

tris

buffer

light microscope

carries information to your eye; place specimen in the path of the beam of light. Light paces through lenses, until reaches eye.

why is regular passaging needed for cell culture

cells need nutrients and room to grow

making tagged antibodies for every antigen is expensive so;

cheaper to have untagged primary antibody to recognize antigen and tagged secondary antibody to recognize all primary antibodies

positive control

colchicine; depolymerization of microtubules

what does the PBS allow in cell culture

constant pH and remove media

if something is turning clear, will the absorbance increase or decrease

decrease

SDS

detergent

2 functions of SDS detergent in SDS-PAGE

disrupt membranes and denature proteins sticks to proteins to change charge

function of heat in SDS-PAGE

disrupts all 4 types of interactions between R-groups and hydrogen bonds involved in alpha helices and beta pleated sheets (secondary)

permeabilization

dissolve cell membrane allow larger dye molecules to enter inside the cell

function of glutamine in medium

energy source

control variable

factor kept constant

why laminar flow hood

filters and sterilizes air while running

steps involved with immunoflourescence

fixation permeabilization blocking antibody exposure mounting

nucleus

genetic material

antibodies

glow green; helps see microtubules and detect apoptosis

g

gravity at sea level

function of BSA in medium

growth factors

primary antibody

has a specific target antigen

first step of differential centrifugation

heavy debri (unbroken cells and walls) separated by short, low speed spin

purpose of loading control

indicate equal loading of samples across all wells and proper transfer of proteins to membrane

sample preparation for western blot

isolate appropriate protein fraction by centrifuge proteins denatured and coated with SDS

fixation

killing cells but keeping them in life like state

standard curve x-axis

known amount

type of hood used for cell culture

laminar flow hood

western blot

looking at specific proteins (antibodies bind to specific antigens)

nucleolus

makes ribosomes

function of glycerol in SDS-PAGE

makes samples dense enough to stay in wells

what is left in supernatant after second step of differential centrifugation

membrane fragments and lower-density organelles

object of SDS-PAGE

minimize difference between proteins so electrophoretic mobility depends on molecular weight

when centrifuging, more dense compounds will

move faster and end up closer to bottom

heavier organelles

move more quickly; not needed to be spun at high speed

emission

narrows exactly what wavelengths reach your eye and camera

light organelles

need spun more quickly in order to pellet

mounting

onto slides allow visual under IFM

2 functions of DTT reducing agent in SDS-PAGE

oxidizes proteins breaks disulfide bridges into 2 separate sulfhydryl groups

environmental factors that influence enzyme function

pH temperature concentration presence of inhibitors/activators

chloroplast

photosynthesis

what does PAGE stand for

polyacrylamide gel electrophoresis

why is sucrose in chloroplast isolation buffer

prevent chloroplast from bursting

function of penicillin in medium

prevent contanimation

without DTT reducing agent

primary structure would not be linearized quaternary structure would stick together

third step of differential centrifugation

purified chloroplasts broken by osmotic shock; thylakoids pelleted away from chloroplasts

total centrifuge force depends on

quickness of rotor spins radius of rotor

DTT

reducing agent

p value less than .05

reject the null hypothesis; is significant

function of tris in SDS-PAGE

resists change of pH

RPM

revolutions per minute

2 functions of bromophenol blue in SDS-PAGE

runs faster than proteins on gel monitors progress

plasma membrane

separate cytoplasm from inside of cell

with secondary antibody

signal is stronger

why must lid be closed during centrifugation

so things don't fly and hit somebody (wait to open until done spinning)

what does SDS stand for

sodium dodecyl sulfate

function of pyruvate in medium

source of carbon and glucose

2 sections of gel with different densities

stacking and separating gel

vacuole

storage; turgor pressure

microtubules

structure/support; motor proteins; cell cycle

second step of differential centrifugation

supernatant (liquid) spun at longer and higher speed to pellet chloroplasts

transmission electron microscope

thin specimens; 2D image; all details of cytoplasm visible. con: takes a long time to process

Why are the cells incubated at 37°C?

to put the enzymes at their optimal temperature to work at

types of electron microscopes

transmission and scanning

how are the cells released

trypsin

standard curve y-axis

unknown amount (measuring)

electron microscope

uses electrons to generate an image

independent variable

variable that is manipulated

dependent variable

variable that is measured

glycerol

viscous liquid

scanning electron microscope

whole specimen; 3D image; very fine details on surface visible