Lecture 3- Cell Separation/Fractionation and Protein Purification

Question 1

What would be the major steps in purifying a specific protein/receptor from a cell?

Answer 1

1.Cell dissociation
2. Cell separation (FACS, velocity sedimentation, selective surfaces, etc)
3. Cell fractionation- designed to separate organells (differential centrifugation, equilibrium density centrifugation)
4. Protein purification- separation POI from others
5. Functional analysis- based on type of protein, widely diverse tests based on the protein

Question 2

What is cell dissociation?

Answer 2

process of breaking down a cell culture to create a suspension of individual cells

Question 3

What is used for cell dissociation and what do they do?

Answer 3

• EGTA=calcium chelator that compromises calcium dependent cell adhesion molecules
• Protease cleaves proteins (breaks peptide bonds between amino acids in a protein) because other proteins are also holding cells together

Question 4

What types of protease are there and when are they used?

Answer 4

• Trypsin: research only
• Liberase: cell therapy such as pancreatic islets transplantation (edmonton procedure)

Question 5

What is the next step after cell dissociation?

Answer 5

• cell sorting: isolating cell of interest from all other cells
• can be done by FACS, GUAVA, Velocity sedimentation, panning, and veridex

Question 6

What is FACS and how does it work?

Answer 6

• Fluorescence activated cell sorting, flow cytometry
• cells labeled w fluroscent mAb that bind to (recognize) cell of interest
• labeled cells are passed through a narrow nozle and flow through a laser beam (to excite), and emitted light is detected by a series of detectors
• detectors measure the intensity of fluorescence for each marker and apply an electric charge
• charged molecules are sorted based on charge- sorting cells ito distinct populations

Question 7

What is guava used for and how does it work?

Answer 7

• cell separation
• flow cytometry that analyzes fluorescently tagged cells using micocapillary tubing- doesn’t sort like FACS, just counts
• can ask how many cells are healthy and how many are sick

Question 8

Velocity sedimentation

Answer 8

• used in cell sorting
• can separate cells based on size using Ficoll to stabilize the media
• big cells exit first

Question 9

What is panning?

Answer 9

• well plate coated w specific antibodies/ligands that bind to cell surface markers unique to the target cell type
• cells placed into coated dish- those that have the right receptor will bind to the coating, the ones that don’t will remain unattached
• dish washed to remove unbound cells

Question 10

Dynabeads

Answer 10

• inert spherical bead (w iron core) has antibodies added to it
• Magnet used to collect beads in test tube… get the cells that bind to the antibody on the magnet

Question 11

Cell Search by Veridex

Answer 11

• method for detecting low number (1 in a billion) CTCs
• FDA approved
• data can monitor a patient’s tumor burden/load

Question 12

What is cell fractionation?

Answer 12

• used to separate organelles from a cell
• steps include lysing cells (breaking down membranes) and centrifugation

Question 13

What are the different methods of lysing cells and which is best?

Answer 13

• sonification- disrupts cell membranes
• detergents-solubilizes membranes
• trituration 9best

Question 14

What is trituration?

Answer 14

• using a small syringe to pull cell up and down, causing shear stress to separate single organelles (w membrane intact)

Question 15

What is a microsome fraction

Answer 15

• rough microsomes (rough ER chunks w ribosomes)
• smooth microsomes (plasma membrane, smooth ER)

Question 16

What do you do once you have the smooth microsome?

Answer 16

• use detergents to solubulize the membrane so protein is no longer integrated in the membrane
• then dialysis
• then final result (have receptors/POI)

Question 17

Once you have the protein what questions do you need to ask?

Answer 17

• What state do I want the protein? (functional or not)
• What do I know about the protein?