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Biochem 2 (CHEM 441B) > Ch. 5 > Flashcards

Ch. 5 Flashcards

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1
Q

What is protein purification?

A

Multi-step fractionation process to isolate a single protein from a mixture

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2
Q

What are the steps of cell fractionation?

A
  1. Prep cells
    - Lyse cells
    - Protein activity is retained
  2. Centrifuge to get different fractions
    - Separation of organelles
  3. Salting out
  4. Dialysis
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3
Q

What is salting out?

A
  • Used to exploit differences in solubility of target protein relative to other proteins in the fraction
    • Adding increasing amounts of saturated salt solution to protein sample
    • Causes formation of insoluble protein aggregates that are functional when resolubilized
    • Often use (NH4)2SO4
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4
Q

What is dialysis? (in terms of protein purification)

A
  • Used to remove ammonium sulfate from protein sample
    • Uses diffusion to leave protein in buffer of proper ionic strength and pH
    • Pores have to be small enough that salt can exit but protein can’t
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5
Q

How should total protein and specific activity change as you purify?

A

Total protein should decrease as activity increases

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6
Q

What are the different column chromatography techniques?

A
  • Size exclusion/gel filtration
  • Ion exchange
  • Affinity
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7
Q

What is column chromatography?

A

Used to separate proteins based on different physical/chemical interactions between the proteins and column matrix

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8
Q

What is size exclusion/gel filtration chromatography?

A

Separates proteins based on size

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9
Q

What is ion-exchange chromatography?

A

Exploits charge differences between proteins

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10
Q

What is affinity chromatography?

A

Exploits specific binding properties of the target protein to separate it from other cellular proteins that lack this binding function

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11
Q

What is gel electrophoresis?

A

Separates proteins on the basis of charge and size

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12
Q

What is SDS-PAGE?

A

Separates denatured proteins by size (final step in protein purification)
- Used to approximate molecular mass of protein or determine whether purified protein includes more than 1 polypeptide chain

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13
Q

What do SDS and β-mercaptoethanol do in SDS-PAGE?

A
  • SDS adds a negative charge to all the proteins
  • β-mercaptoethanol breaks disulfide bonds
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14
Q

What is isoelectric focusing?

A

Separates proteins on the basis of charge as a function of pH

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15
Q

What is 2D gel electrophoresis?

A

Isoelectric focusing combined with SDS-PAGE
- Based on pI and molecular mass

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16
Q

What are polyclonal antibodies?

A

Heterogeneous mixture of Ig proteins that recognize 1 or more epitopes on antigenic protein

17
Q

What are monoclonal antibodies?

A

Homogeneous Ig species that recognizes 1 epitope on antigenic protein

18
Q

What research methods use antibodies?

A
  • Western blot
  • Affinity chromatography
  • Immunofluorescence
  • Immunoprecipitation and CoIP
  • ELISA
19
Q

What is a Western blot?

A

Used to detect proteins separated by gel electrophoresis
- Uses primary (protein-specific) and secondary (detection) antibodies

20
Q

What is immunofluorescence?

A
  • Used to identify proteins in cells that have been chemically treated in a way that preserves the cell’s architecture
  • After washing, cells can be visualized through fluorescence microscopy
21
Q

What is ELISA?

A
  • Identifies low level antigenic proteins
  • Direct and indirect ELISA
22
Q

What is the Edman degradation?

A

Protein sequencing method based on labeling and cleaving the N-terminal residue without disrupting the rest of the polypeptide chain

23
Q

How is protein cleavage carried out?

A
  • Trypsin: cleaves on C side of lys and arg
  • Chymotrypsin: cleaves on C side of tyr, trp, and phe
  • Cyanogen bromide: cleaves on C side of met
  • S. aureus: cleaves on C side of asp and glu
24
Q

What kind of enzymes are trypsin and chymotrypsin?

A

Proteases

25
Q

What is mass spectrometry?

A

Method of measuring the mass-to-charge ratio of molecules, which is then used to deduce the molecular mass

26
Q

What is X-ray crystallography?

A

Diffraction of X-rays from crystals of biomolecules is used to determine the locations of atoms in the structure

27
Q

What are the pros and cons of X-ray crystallography?

A
  • Pro: protein can be big
  • Con: protein is dead
28
Q

What is NMR spectroscopy?

A

Detects nuclear spin properties of certain atoms (1H, 13C, 15N) to deduce their relative locations

29
Q

What are the pros and cons of NMR spectroscopy?

A
  • Pro: protein is dynamic (not dead yay)
  • Con: protein has to be small

Biochem 2 (CHEM 441B) (23 decks)

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