BIO 311 Exam I: Cell Separation & Protein Techniques

Cell Separation

Separating cells in a tissue sample

Cell Fractionation

Take a separated cell and separate organelles

Protein Purification

Separate the POI

Question: How can purify the insulin receptor from Cell X

1) Cell Dissociation: Separate Cell X from other cell types

2) Cell Sorting: Discard other cells and keep Cell X

3) Cell Fractionation: Break the cell down into its organelles/ Acquire the desired organelle

4) Protein Extraction: Go from a microsome to an fluid enriched with POI

5) Protein separation/enrichment: Get our proteins (almost) completely isolated

Cell Dissociation

- EGTA: Calcium sponge that comprimises Ca dependant cell adhesion molecules

AND

- Protease: Cleaves the proteins that attach cells (Liberase or Trypsin)

Cell Sorting

Many Options:

- Fluorescence Activated Cell Sorting

- GUAVA

- Velocity Sedimentation

- Panning

- Veridex (CTC)

Fluorescence Activated Cell Sorting

- A fluorochrome is attached to a mAb that recognizes Cell X

- Then all the cells in the batch are run between two charged plates that sort the cells into different tubes based on the fluorescent molecule attached

GUAVA

Doesn't truly sort cells, but does count the number of cell types using fluorochromes

Velocity Sedimentation

- Separates cells based on size using gravity

- Makes use of a ficoll gradient that stabilizes the media

Panning

- Cells are placed on a plate with ligands attached

- The receptors on the cells attach to the ligands

OR (more commonly)

- Inert dynabead with iron core and receptors attached

- Cell attaches and is pulled out with a magnet

Veridex

- Cell search for CTCs

- mAb anti-EpCAM beinds to EpCAM which almost only exists on CTCs

- An iron bead with mAb anti-EpCAM binds to CTCs and counts them

- Allows you to count the number of CTCs and estimate cancer burdern

Cell Fractionation

Steps:

1) Lyse the Cells

- sonication

- detergent

- Trituation

2) Differential Centrifugation

3) Rate Zonal/Equilibrium Density/ Density Gradient

Sonication

Disruption of cell membrane using sound waves

detergent

solubilizes membranes

Trituation

small syringe moves cell up and down, breaking down the cell into single organelles

Differential Centrifugation

- solution with lysed cell is spun numerous times

- Each time it is spun the heaviest stuff moves to the bottom

- The top liquid or supernatant can be spun again until you reach the desired item size

- In our case we want "microsome fraction" which contains rough microsomes from RER and smooth microsomes from the membrane

Rate Zonal/Equilibrium Density/Density Gradient

- The microsome fraction is placed in a container with a ficol gradient and spun

- The objects then reach a point in the ficoll gradient that they are equal in density to and stay there allowing for extraction

- In the case of the microsome fraction you have the smooth and rough microsomes separating

Protein Extraction

- We now have smooth microsomes from X but we don't have the receptor we want yet

- Detergent is used to break down the phospholipid outised of microsome and surround POI

- Ends with a liquid enriched with POI

Protein Separation/Enrichment

- This step depends on how we want the protein to end up

- Functional?

- Also factors like the size and charge of the protein contribute to this step

- Liquid Chromatography

Liquid Chromatography

Various methods of separating proteins to enrich the sample

- Ion Exchange Chromatography

- Gel Filtration/sizing column

- Affinity chromatography

Ion Exchange chromatography

- Separates proteins based on charge

- Charged beads are placed with the liquid soup of proteins

- Desired proteins will either stick or not

- Most proteins are negative

Gel Filtration

- Used if the POI has a much different size than other proteins

- Pourous gel beads are used and smaller proteins get stuck in the bead while biggers once go past it

Affinity Chromatography

- Separates proteins based on interaction with another protein

- A bead is covered with ligands that react to the receptor

- The POI attaches and everything else leaves

- The solution is then salted to get the POIs to come off then desalted to return the original POI shape

Native Gel Electrophoresis

- Proteins are run in a gel in their native state

- A power supply creates a charge difference that moves proteins

- Distance traveled = weight

- Benefit: The gel can be cut out and examined directly since proteins are in their original shape

SDS Gel Electrophoresis

- Does not preserve native state

- Has better resolution that native

Steps:

1) Boil and denature protein sample

2) Add urea to disrupt hydrogen bonds and B-mercaptoethanol to hydrolize disulfide bonds

3) Add SDS which is a detergent

Isoelectric Focusing

- gel has a pH gradient

- proteins vibrate until they reach their desired pH point were they are electrically neutral

2D Gel electrophoresis

- Best resolution

- Starts with isoelectric focusing then does SDS

Western Blots

- Gel eletrophoresis

- Uses mAbs to indentify POI

- Allows you to figure out molecular weight of POI and compare it to other POIs

Steps:

1) Run an SDS gel

2) Transfer proteins out of gel onto transfer paper so that mAbs can access them

3) enzyme attached to mAb attached to POI will react with substrate and indicate position

Photoreactive amino acids w/Western Blots

- Amino acids that respond to UV light and will cause proteins to make covalent bonds with their ligand/receptor

- A western blot is done with UV light hit proteins

- The UV proteins are much heavier because they are bound together

SELDI-TOF/MALDI-TOF

- Uses a laser and proteins on a chip

- Laser hits proteins and sends them flying

- flying velocity=protein size

- chip holds proteins in place with a charge difference or mAb

Protein A Immunoprecipitation

- Protein A can bind to all mAbs

- A bead is covered in protein A then mAbs specific to a POI bind to protein A

- POIs then bind to the mAb

- Spin pellet in centrifuge to get POIs off

Autoradiography

- Uses radioactive probe to measure cell activity

- Can be paried with gel electrophoresis

Cell Culture

- A growth of cells in an artificial environment

George Gey

- Established HeLa cells in culture but couldn't get them to divide

- Made HeLa cells

Leonard Hayflick

- Made somatic cells in culture but couldn't get them to divide more than 25-50 times

Power of Cell Culture

- Allows you to observe cells in controlled environment

Limits of Cell Culture

- Genetic drift

- Phenotype change

MDCK Cells

- Kidney cells that don't function correctly in vitro

- After going through cell culture insent-microporous device they get polarized and start funcitoning like kidney cells

Hepatocytes

Issues:

- Don't divide in vitro

- Lose filtration functoin over time (Cyt p450)

Cell Strain

- Freshly isolated from a primary tissue

- 25-50 divisions at most before apoptosis

- Very similar to in vivo but will die soon

Cell Line

- Makes ue of immortal cells

- Embryonic: Embryos that continue dividing

- Cancer Cells: Tumors that also keep growing

- Good but cancer cells don't accurately represent healthy cells in vivo

- Subject to genetic drift

Telomerase Immortal Cell Line

- Cell line that has ben made immortal by being trasnfected wiht telomerase

Cell Media Requirements

- Serum Supplemented Media

- Serum Free Media

Serum Supplemented Media

- Made with 10% fetal calf serum

- We know cells need growth factors but we don't know which ones

- Sucks because its expensive and also may contain weird bovine pathogens and animal proteins that cause problems in cell therapy

Serum Free Media

- As we gain more knowledge we are able to put together a media that has as many growth factors as necessary without having to add animal stuff

Growth Substrates

- Cell Culture Dishes

- Microcarrier Beads

- Rollerbottles

- Tube

Cell Culture Dish

- Can carry around 1536 cells

Microcarrier beads

- Used in batch cultures for very large amomunts

- Cells grow on microcarrier bead with mAbs to stick to bead

Rollerbottles

- Cells grow on the wall of the bottle while they roll, occasionally covering the cells in media

Tube

- Good for vascular tissue since it replicates the in vivo conditions

- Cell culture inserts

- Replicates endothelial cells in transcytosis

Transcytosis

- The passing of antibodies across an endothelial layer

- Occurs between the blood and milk via mammary cells

Tissue engineering

Use of cells to generate lab grown constructs

Three sections of Tissue Engineering

- Cells, Materials and Tissue Architecture

ELAD

- Cartridges of liver cells that are grown with C3A cancer hepatoma cells which have good cyto P450 levels