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PATH2211 > 17 - Protein Detection > Flashcards

17 - Protein Detection Flashcards

1
Q

C-reactive protein

A

Activates immune system. Triggered by cytokines and proteins

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2
Q

Why detect proteins

A

As a marker of inflammation and to detect pathology (e.g. myocardial infarction).

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3
Q

4 levels of protein structure

A

Primary (amino acids), Secondary (beta sheet and alpha helix), Tertiary (polypeptide chain) and Quaternary (assembled subunits)

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4
Q

Which proteins detected to indicate myocardial infarction

A

Contractile proteins (Troponin 1 and T)

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5
Q

Chromatography column

A

Solid porous matrix (“stationary phase”)

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6
Q

Mobile phase

A

Proteins sample that flows through chromatography column

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7
Q

Which proteins elute first

A

Proteins that move quickly (interact less well with column)

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8
Q

Which proteins elute last

A

Proteins that move slowly (interact more with column)

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9
Q

Fractionation

A

Collection of effluent at different stages. Different fractions have different properties

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10
Q

Eluent

A

Substance that goes through column chromatography

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11
Q

Effluent

A

Substance that comes out of column chromatography

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12
Q

Size exclusion chromatography

A

Column made of porous beads. Larger proteins do not adhere to column (therefore move quickly and elute earlier). Small proteins adhere to column (therefore move slowly and elute later)

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13
Q

How do proteins have different charges

A

The functional groups of individual amino acids confer an overall charge to the protein

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14
Q

What can change the charge on the AA side chain

A

pH. Acids lose/gain H+, bases lose/gain OH-

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15
Q

Ion exchange chromatography

A

Column is made of charged gel beads. If stationary phase is negatively charged, proteins with negative charge will move faster and elute earlier.

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16
Q

Issue with ion exchange chromatography

A

proteins with different charge to stationary phase will not elute out and therefore become stuck

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17
Q

How to fix issue with ion exchange chromatography

A

Add salt (charged ions). Ions outcompete protein for binding to column

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18
Q

Cation exchange

A

negative column, positive samples of interest

19
Q

Anion exchange

A

Positive column, negative samples of interest

20
Q

Affinity chromatography

A

Column made of beads with a molecule (ligand) to which the target binds. Target protein binds, non target are eluted.

21
Q

how to elute target protein in affinity chromatography

A

With solution of ligand. Target protein will bind to free floating ligands instead of those attached to column

22
Q

Other forms of affinity chromatography

A

Antibody - antigen
Avidin - biotin
Nickel - Histidine tag

23
Q

How to elute affinity chromatography that doesn’t contain ligands

A

high pH dentures the proteins so they no longer bind

24
Q

SDS-Page

A

SDS (detergent, denatures proteins, gives uniform -‘ve charge), polyacrylamide plastic (same function as agarose but seperate much smaller molecules). Larger proteins migrate less far

25
Q

advantages of SDS-PAGE

A

Quick, easy and inexpensive

26
Q

Disadvantages of SDS-page

A

Visualise proteins with stain (small amount detected), does not identify proteins, many proteins have similar mass, unable to tell how many proteins a band shows

27
Q

Charge when pH is less than pKa

A

positive (protonated)

28
Q

Charge when pH = pKa

A

No charge

29
Q

charge when pH is greater than pKA

A

Negative charge (deprotonated)

30
Q

Isoelectric focusing

A

Separates proteins by isoelectric point. Protein only moves through gel when it is charged. Reaches point in strip where pH = pI (protein has no charge, will not migrate further)

31
Q

2D electrophoresis

A

Separates proteins by isoelectric point and size. Solves problem of co migration in SDS-PAGE

32
Q

Western blots (immunoblots)

A

Transfer proteins from polyacrylamide gel to a membrane. Allows immobilisation of the protein which allows antibodies to interact with the protein

33
Q

Components of Western blots

A
  • Primary antibody recognises antigen
  • Secondary antibody recognises primary antibody (enzyme conjugated)
  • Enzyme allows detection (catalyses reaction to produce luminescence)
34
Q

Enzyme Linked Immunosorbent Assay (ELISA)

A

Use antibodies to recognise specific antigen (protein of interest)

35
Q

Direct ELISA

A
  • Proteins are immobilised to a plate
  • Antibody binds to target protein and is conjugated to an enzyme
  • Enzyme produces colour product
  • Less specific
36
Q

Indirect ELISA

A
  • Primary antibody binds to target protein
  • Secondary antibody binds to primary antibody
  • Secondary antibody is conjugated to an enzyme which produces a coloured product
37
Q

Sandwich ELISA

A
  • Capture antibody bonded to plate and binds to target protein
  • Primary antibody binds a different epitope on the target protein
  • Secondary antibody binds to primary antibody, conjugated to enzyme to detection
38
Q

Which protein side chains absorb light

A

Aromatic group

39
Q

High Performance Liquid Chromatography

A
  • Column usually C18 (silica) which is hydrophobic.
  • Sample dissolved in water and non polar solvent.
  • Hydrophobic proteins in sample will bind to C18.
  • Hydrophilic proteins elute.
  • Change solvent over time to be more hydrophobic to elute the “stuck” hydrophobic proteins
  • Achieves gradient of elution (hydrophilic –> hydrophobic)
40
Q

Mass spectrometry

A
  • Measure mass of compounds with great accuracy.
  • Compound ionised
  • Charge attracts it to detector
  • Arrive at detector at different times based on mass to charge ratio
  • Small mass to charge ratio and faster, large mass to charge ratio slower
  • Also fragments peptides and measures products of fragmentation
41
Q

Mass fingerprint

A

Two different proteins with the same mass will produce different peptides

42
Q

How is protein prepared for mass spectrometry

A

Digested with proteolytic enzyme

43
Q

Advantages of mass spectrometry

A

Generate huge amounts of detailed data

44
Q

Disadvantages of mass spectrometry

A
  • Expensive, complex machines needed
  • Data can be difficult to interpret
  • Difficult sample preparation

PATH2211 (21 decks)

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