Lecture 3 Flashcards
Quantification of cells expressing 2 different cell surface markers by FACS
- two different colours of fluorescence will be observed and recorded with each dot representing a single cell
- e..g. We are looking for the cells that express both proteins on their outermmebranes
Cell cycle analysis by facs
- measuring the cells that have fluorescence using FACS
- blue fluorescence is indicative of the amount of DNA found in the cells
Where are most cells found in cell cycle analysis by FAC
- in G1 which occurs before ell undergoes replication
what stage willl have the highest intensity of hoechst stain fluorescence
- cells that have replicated their DNA but not fully divided
what does the cell cycle analysis with FACS allow us to do
quantify cells at different stages of cell cycle
how to disrupt plasma membrane
- mechanical homogenizatoin
- sonication
- pressure
- non ionic detergents e.g. triton x-100
- placing cells in hypotonic solution
sonicaiton
ultrasound
pressure
forcing cells through a small diameter tube to disrupt the plasma maebrane
non ionic detegents
chemicals integrate into plasma mmebmrane causing its dissociation
hypotonic solution
causes cell lyss
bench top centriguation
- low speed
ultracentrifuges
- spinning at high sepeds
how is heat created in centriguation
when too spins around and around, takes stuff and forces it to bottom, spins so high interacts with oxygen molecules to create insnare heat
why is it important to ensure balanced centriguation
- so that the weigh t is distributed and not become a projectile with the rotor breaking off its axis
differential centrifugation
increasing centrifugal force (gravity) to isolate organelles based on their mass
600g
seperates nuclei
15 000 g
operates mitochondria, chloroplast, lysosome, peroxisomes
100000g
PM, micrsomal fraction (fragments of ER) and large plyribsoomes
300 000
ribosomal subunits, small polyribosomes, then can pour out soluble part of cytoplasm Icytosol)
how does differential centriguation work
-successive series of centrifugal steps, increasing the force at each step startingf fro low and going higher, get progressively smaller stuff in the pellet
how do isolate a specific organelle from the pellet
- use eq desnity gradient centirguation
eq density gradient centriguation
- seperation based on ensity
- homogenate is applied to a gradient of sucrose
- apply a centreigual face
-organelles will move down sucrose gradient and stop when they reach their equivalent density
order of density for peroxisomes, mitochondria and lysosomes
peroxisomes (most dense), mito, lysoosmes (least dense)
non ionic detergents
- disrupt the lipid bilayer r only
ionic detergents
- dentature and disrupt ionic and hydrogen bonds
detergents are ___ molecules
amphipathic
amphipathic
both hydrophobic and hydroponics components
which type of detergent should u use if u just want to disrupt the lipid bilayer
non ionic
which detergent should u use if u want to denature and disrupt protein-protein interactions
ionic
SDS page
- electoprhoertic separation of proteins
SDS detergent
- Ionic detergent, immerse proteins with this, disrupts 3D structure and bind to Phobic portions within a protein and give a net negative charge to protein bc of negative sulphate anion
charge to mass ratio
all of the proteins are forced into this negatively charged conformation
how to see and quantity the proteins after SDS page
using a chemical stain.dye e,.g, coomassie brilliant blue or silver
coomassie stain
- blue
detecting a specific protein after SDS page
- antibody against the specific protein is added tot he gel
- transfer the proteins to the gel for better visualization/pentration
- incubate with the pimriary antibody which oldy bind to the proteins that are at a certain molecular weight
- use secondary antibody that is bound to horse radish peroxidase
- put inn dark box to capture light or x ray film to get an idea of the amount of a specific protein
how does horse radish peroxidase help in the illumination of specific proteins
- it is bound to the secondary antibody, and reacts with substrate luminal to take it form non luminescent to luminescent
