Chapter 8: Analyzing Cells Flashcards

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1
Q

Method to study protein encoded in RNA

A

use bacteria (they’re simple)

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2
Q

Method to study function of organelles/intracellular functions

A

use eukaryotic cells

don’t need something more complex, such as context in a tissue

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3
Q

cells can be isolated from tissues

A

most realistic

1) disrupt extracellular matrix and cell-cell junctions (use proteolytic enzymes to digest proteins and chelator agents)
2) tease apart tissue into cells by gentle agitation

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4
Q

methods to break open cells (don’t destroy the inside!)

A

high frequency sound, detergent (pokes holes in membrane), force cells through small tight spaces so they burst, glass vessel and pestle to shear open

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5
Q

what do you end up with after breaking open cells?

A

a homogenate

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6
Q

homogenate

A

mix of membrane components, cytoplasm, proteins

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7
Q

cells can be grown in culture

A

aka in vitro (in glass)

can culture plant and animal cells

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8
Q

purifying protein step one

A

extraction from inside the cell (break cells open)

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9
Q

centrifugation

A

rotates cell extracts at high speeds

separates them by size and density

impure, multiple centrifugations can improve purity

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10
Q

centrifugation where do larger objects go

A

large object = most centrifugal force = pellet at bottom

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11
Q

low speed centrifugation

A

pellet contains whole cells, nuclei, cytoskeletons

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12
Q

medium speed centrifugations

A

pellet contains mitochondria, lysosomes, peroxisomes

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13
Q

high speed centrifugation

A

pellet contains microsomes, small vesicles

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14
Q

very-high speed centrifugation

A

pellet contains ribosomes, viruses, large macromolecules

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15
Q

supenatant

A

liquid above pellet

contains lighter molecules

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16
Q

microsomes

A

resealed vesicles formed from fragments of rough and smooth ER

17
Q

column chromatography

A

separates by interaction w/ matrix

18
Q

ion exchange chromatography

A

separates on charge
resin is positive, positive flows through
can elute with diff concentrations of salt

19
Q

hydrophobic chromatography

A

hydrophobicity

column contains beads with hydrophobic chains, retard hydrophobic

20
Q

gel-filtration chromatography

A

size
large go through first, small get stuck in pores of beads
helps estimate size

21
Q

affinity chromatography

A

ability to bind to particular small molecules or to other macromolecules

EX: bead covered in polysaccharide, enzyme that operates on it will be retained

later can be washed off in pure form

EX: antibodies

22
Q

high-performance liquid chromatography (HPLC)

A

silica based resin
separates even at fast flow rates

great choice!

23
Q

protein purification by chromatography requires:

A

multiple steps in succession (usually)

a functional assay to follow purification so you know what fractions to keep