methods for protein study

Question 1

what is a cell culture?

Answer 1

a propagation of cells in a culture dish outside the organism

Question 2

what are the advantages of a cell culture?

Answer 2

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

Question 3

why is it important to study the function of gene regulatory proteins?

Answer 3

a single gene regulatory protein can permanently turn on a cascade of gene regulatory proteins and cell-cell interaction

Question 4

what are primary cultures?

Answer 4

cells derived from tissues

Question 5

what are the advantages and disadvantages of primary cultures?

Answer 5

advantages: best experimental model of in vivo cells
disadvantages: cells have a limited lifespan and cease to grow after a few divisions

Question 6

what are cell lines?

Answer 6

cells derived from primary cells or tumours

Question 7

how are cell lines created from primary cells?

Answer 7

treatment with radiation, chemical carcinogens, tumour causing viruses

Question 8

what are the characteristics of cell lines (transformed cells)?

Answer 8

• immortality due to lack of external growth factor

- altered morphology; loss of growth control, lack of contact inhibition

Question 9

what are the advantages and disadvantages of cell lines?

Answer 9

adv: cells continue to grow and divide indefinitely in appropriate culture conditions
disadv: cells are different to normal celsl

Question 10

what are telomeres?

Answer 10

caps at the end of DNA strands made of hexonucleotide repeats, which prevent chromosomes from unravelling

Question 11

what is the end replication problem?

Answer 11

telomeres shorten every S phase of replication. telomeres get shorter and cell function decreases, this limits the number of cell divisions in primary cells

Question 12

what conditions/media do cells require to grow in culture?

Answer 12

• 37 degrees, humidified atm, 5% CO2
• amino acids
• vitamins

Question 13

what are the ways in which you can make a cell lysate >

Answer 13

• hypotonic lysis
• homogenisation: cells disrupted by shear forces or grinding
• freeze thawing
• biological detergents

Question 14

what is the composition of a typical mammalian cell lysis buffer?

Answer 14

• 0.01M Tris-HCl
• 0.15M NaCl
• 1% w/v detergent
• protease inhibitors
• MgCl2
• Phosphatase inhibitors

Question 15

define biological detergent

Answer 15

small amphipathic molecules that form micelles in water

Question 16

what is an ionic detergent?

Answer 16

e.g. SDS; strong detergent that denatures protein by binding to internal hydrophobic core

Question 17

what is a non-ionic detergent?

Answer 17

e.g. Triton-X100; a mild detergent that binds only to the membrane spanning domain of the transmembrane proteins

Question 18

explain how a detergent solubilises a protein?

Answer 18

detergent binds to hydrophobic regions of a membrane protein, displacing the lipid molecules. the lipid bilayer is disrupted and the cell contents are released

Question 19

list the different forms of chromatography that can be used to separate proteins?

Answer 19

• ion exchange chromatography
• hydrophobic chromatography
• gel filtration chromatography
• affinity chromatography

Question 20

what is ion-exchange chromatography?

Answer 20

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

Question 21

what is hydrophobic chromatography?

Answer 21

proteins separated according to their hydrophobicity. column is packed with beads that have hydrophobic side chains; selectively retarding proteins with exposed hydrophobic regions

Question 22

what is gel-filtration chromatography?

Answer 22

proteins are separated according to their size via tiny porous beads

Question 23

what is 1D SDS-PAGE?

Answer 23

separates proteins according to their size only

Question 24

what is 2D SDS-PAGE?

Answer 24

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

Question 25

what is western blotting?

Answer 25

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