methods for protein study Flashcards
what is a cell culture?
a propagation of cells in a culture dish outside the organism
what are the advantages of a cell culture?
1- single, well defined cell type
2- large quantity of cells
3 - environment of the cell can be controlled
4- it is easier to study cell function in vitro
why is it important to study the function of gene regulatory proteins?
a single gene regulatory protein can permanently turn on a cascade of gene regulatory proteins and cell-cell interaction
what are primary cultures?
cells derived from tissues
what are the advantages and disadvantages of primary cultures?
advantages: best experimental model of in vivo cells
disadvantages: cells have a limited lifespan and cease to grow after a few divisions
what are cell lines?
cells derived from primary cells or tumours
how are cell lines created from primary cells?
treatment with radiation, chemical carcinogens, tumour causing viruses
what are the characteristics of cell lines (transformed cells)?
- immortality due to lack of external growth factor
- altered morphology; loss of growth control, lack of contact inhibition
what are the advantages and disadvantages of cell lines?
adv: cells continue to grow and divide indefinitely in appropriate culture conditions
disadv: cells are different to normal celsl
what are telomeres?
caps at the end of DNA strands made of hexonucleotide repeats, which prevent chromosomes from unravelling
what is the end replication problem?
telomeres shorten every S phase of replication. telomeres get shorter and cell function decreases, this limits the number of cell divisions in primary cells
what conditions/media do cells require to grow in culture?
- 37 degrees, humidified atm, 5% CO2
- amino acids
- vitamins
what are the ways in which you can make a cell lysate >
- hypotonic lysis
- homogenisation: cells disrupted by shear forces or grinding
- freeze thawing
- biological detergents
what is the composition of a typical mammalian cell lysis buffer?
- 0.01M Tris-HCl
- 0.15M NaCl
- 1% w/v detergent
- protease inhibitors
- MgCl2
- Phosphatase inhibitors
define biological detergent
small amphipathic molecules that form micelles in water
what is an ionic detergent?
e.g. SDS; strong detergent that denatures protein by binding to internal hydrophobic core
what is a non-ionic detergent?
e.g. Triton-X100; a mild detergent that binds only to the membrane spanning domain of the transmembrane proteins
explain how a detergent solubilises a protein?
detergent binds to hydrophobic regions of a membrane protein, displacing the lipid molecules. the lipid bilayer is disrupted and the cell contents are released
list the different forms of chromatography that can be used to separate proteins?
- ion exchange chromatography
- hydrophobic chromatography
- gel filtration chromatography
- affinity chromatography
what is ion-exchange chromatography?
the column is packed with small beads that carry a positive or negative charge, proteins are fractionated according to the arrangement of charges on their surface
what is hydrophobic chromatography?
proteins separated according to their hydrophobicity. column is packed with beads that have hydrophobic side chains; selectively retarding proteins with exposed hydrophobic regions
what is gel-filtration chromatography?
proteins are separated according to their size via tiny porous beads
what is 1D SDS-PAGE?
separates proteins according to their size only
what is 2D SDS-PAGE?
proteins are first separated according to their charge by dissolving in a sample of non-ionic detergent, b-mercaptoethanol and urea to solubilise, denature and dissociate polypeptide chains but leave intrinsic charges unchanged
what is western blotting?
an electric current is applied to the gel so separated proteins transfer to the membrane in the same pattern as they separated on the SDS-PAGE gel
