methods for analysing cell proteins

Question 1

what is a cell culture?

Answer 1

propagation of cells in a culture dish outside the organism

Question 2

what are the advantages of cell cultures?

Answer 2

1- produce a single, well-defined cell type
2- produces a large quantity of cells
3- the environment of the cells can be controlled
4- it is easier to study cell function in vitro

Question 3

what is a primary culture?

Answer 3

cells derived from tissues

Question 4

what are the advantages and disadvantages of primary cultures?

Answer 4

• best experimental model of in vivo cells

- limited lifespan

Question 5

what are cell lines?

Answer 5

cells derived from primary cells or tumours

Question 6

how can cell lines be created from primary cells?

Answer 6

treatment with radiation, chemical carcinogens, or tumour viruses

Question 7

what is different about transformed cells?

Answer 7

they have altered morphology and will exhibit loss of growth control, loss of contact inhibition

Question 8

what are the advantages and disadvantages of transformed cells?

Answer 8

• will continue to grow and divide indefinitely as long as appropriate culture conditions are maintained
• different to normal cells

Question 9

what are telomeres and what are they for?

Answer 9

• caps at the end of DNA strands made of hexonucleotide repeats
• prevents the chromosomes from unravelling

Question 10

what is meant by the end replication problem?

Answer 10

telomeres shorten with every replication and cell function decreases. limits the number of cell divisions in primary cells

Question 11

what culture conditions are required to grow cells?

Answer 11

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

Question 12

what different techniques can be used to make a cell lysate?

Answer 12

• use a hypotonic buffer
• homogenisation
• sonication
• freeze thawing
• biological detergents

Question 13

what is the purpose of the techniques which are used to make a cell lysate?

Answer 13

break up membranes within the cell, which form fragments and immediately reseal to form closed vesicles

Question 14

what is the composition of a mammalian cell lysis buffer?

Answer 14

0.01M Tris-HCl pH 7.4
0.15M NaCl
1% w/v detergent
protease inhibitors
MgCl2
phosphatase inhibitors

Question 15

what are biological detergents?

Answer 15

small amphipathic molecules that form micelles in water

Question 16

give an example of an ionic detergent, how does it work?

Answer 16

SDS - strong, denatures proteins by binding to their internal hydrophobic cores, inactivating the protein

Question 17

give an example of a non-ionic detergent, how does it work?

Answer 17

Triton-X100. mind, only binds to the membrane spanning domain of the transmembrane proteins, allows the protein to remain active

Question 18

what is ion-exchange chromatography?

Answer 18

where proteins are separated according to charge. a column is packed with small beads that carry a positive or negative charge. proteins fractionated according to the arrangement of charges on the surface

Question 19

what is hydrophobic chromatography?

Answer 19

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

Question 20

what is gel filtration chromatography?

Answer 20

where proteins are separated according to their size via tiny porous beads that are able to filter through

Question 21

what is affinity chromatography?

Answer 21

separation of a protein based on its ability to bind a specific molecule immobilised on an insoluble matrix

Question 22

what is the principle behind affinity chromatography?

Answer 22

if a substrate is covalently coupled to an inert matrix, e.g. polysaccharide bead, the enzyme that operates on the substrate will be specifically retained by the matrix and can be eluted in pure form

Question 23

what is one dimensional SDS-PAGE?

Answer 23

separation of proteins according to their size only

Question 24

what is 2D-PAGE?

Answer 24

proteins are separated first according to their charge by dissolving in a sample of non-ionic detergent, b-mercaptoethanol and urea

Question 25

how does 2D PAGE work?

Answer 25

the solution solubilises, denatures and dissociates the polypeptide chains, but leaves their intrinsic charges unchanged. they are then separated according to their size using a pH gradient and isoelectric focusing

Question 26

which techniques can be used to identify a protein following SDS-PAGE?

Answer 26

western blotting

mass spectrometry

Question 27

how does western blotting work?

Answer 27

an electric current is applied to the gel, so the separated proteins transfer through it and onto the membrane in the same pattern as they separated onto the SDS-PAGE gel.