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ESA1 - Molecules, Genes And Disease > Molecular Techniques & Diagnosis of Proteins > Flashcards

Molecular Techniques & Diagnosis of Proteins Flashcards

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1
Q

What is serum protein electrophoresis?

A

Test that examines specific serum proteins in blood called GLOBULINS

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2
Q

How many major bands would you expect in the gel for serum protein electrophoresis? What do they represent?

A

5 major bands representing the globulins in the blood:

  • ALBUMIN
  • α-1-globulin
  • α-2-globulin
  • β-globulin
  • γ-globulin
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3
Q

How would an abundance of a serum protein be identified on the gel?

A

DARKER BAND

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4
Q

What stain is normally used in serum protein electrophoresis?

A
  • PONCEAU S stain

- Stains bands BLUE

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5
Q

Explain how protein gel electrophoresis allows separation on the basis of size AND charge

A
  • Size - smaller proteins travel and migrate through gel faster than larger proteins
  • Charge - proteins have a range of charges at physiological pH due to their ISOELECTRIC point, so -ve proteins will move towards anode and +ve proteins will move towards cathode
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6
Q

What does the speed of travel of a protein through gel depend on?

A
  • Size - smaller proteins will travel faster than larger proteins
  • Charge - proteins with a higher charge will be more attracted to the anode or cathode than proteins which are uncharged
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6
Q

Explain the clinical significance of serum protein electrophoresis

A
  • Can identify the serum protein abundances in normal blood and compare this to the serum protein levels in diseased state
  • Change in serum protein levels may be used diagnostically
  • e.g. Decreased serum albumin and increased γ-globulin may indicate multiple myeolma
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7
Q

What method could you use to separate proteins purely on the basis of SIZE?

A

SDS-PAGE

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8
Q

Why does SDS-PAGE use unfolded proteins?

A
  • Need proteins to have a singular charge

- Folded proteins have an intrinsic charge

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9
Q

Explain the action of SDS and β-ME in SDS-PAGE

A
  • SDS denatures protein by breaking intermolecular forces within tertiary and secondary structure
  • SDS binds to primary structure in specific places spreading a UNIFORM NEGATIVE CHARGE
  • β-ME breaks disulphide bonds within tertiary structure
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10
Q

How does SDS-PAGE allow us to identify the presence of proteins?

A
  • Can identify size and use database to identify proteins of that size
  • Can identify unknown proteins by comparing them to known proteins adjacent in the gel and estimating their size
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11
Q

Explain how ISOELECTRIC FOCUSING can be used to separate proteins purely on the basis of CHARGE

A
  • Each protein has a different isoelectric point (pI) which states the pH at which it has no overall net charge
  • Gel in cylinder has electric field which establishes a pH gradient
  • Proteins migrate towards anode or cathode depending on charge
  • When protein reaches its pI in the pH gradient it stops migrating and is stained to form a band
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12
Q

What is 2D-PAGE used for?

A
  • Allows the separation of complex protein mixtures

- Important for diagnosing disease states in different tissues

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13
Q

Explain the process of 2D-

PAGE in the identification of proteins

A
  • Isoelectric focusing used to separate proteins purely on basis of CHARGE (bands form where proteins have similar pI)
  • SDS then used to separate proteins in each band on the basis of SIZE (can identify how many proteins have similar charge AND size)
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14
Q

Briefly explain how proteins can be identified using PROTEOMICS

A
  • Digest protein with restriction enzyme e.g. Trypsin (cuts at Lys and Arg residues)
  • MASS SPECTROMETRY
  • Use database of peptide sizes for known proteins to identify UNKNOWN proteins
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15
Q

What is the difference between proteomics and molecular diagnosis?

A
  • PROTEOMICS is the analysis of all proteins expressed in a genome
  • MOLECULAR DIAGNOSIS is the analysis of a single purified protein
16
Q

Give 2 examples of enzymes which can perform specific proteolytic cleavage

A
  • TRYPSIN - specifically cleaves at Lys and Arg residues
  • STAPHYLOCOCCAL PROTEASE - specifically cleaves at Asp and Glu residues
  • Cleavage involves breaking of peptide bond within amino acid sequence
17
Q

What is an EPITOPE?

A
  • Few amino acids present on an antigen

- Antibodies can recognise specific epitomes on antigens and bind to them complementary

18
Q

What are POLYCLONAL antibodies?

A
  • Multiple different antibodies that are specific to 1 antigen which contains multiple epitopes
  • Produced by many B lymphocytes
20
Q

What are MONOCLONAL antibodies?

A
  • Identical antibodies which are specific to 1 antigen which contains 1 epitope
  • Produced from 1 B lymphocyte
21
Q

Explain how you would isolate monoclonal antibodies

A
  • Inject animal with specific antigen which forms an immune response
  • Isolate spleen cells and combine with myeloma cells to prolong life so cells continue to divide
  • Lots of antibodies of 1 particular type are produced; these can then be isolated by injecting them back into animal and growing tumour OR growing cells in a mass culture
22
Q

What is WESTERN BLOTTING used for?

A
  • Detection of proteins on SDS-PAGE gel using antibodies

- Can detect specific proteins present in mixtures

23
Q

Explain how polyclonal antibodies can be isolated

A
  • Inject animal with known antigen 3-4 times over a 2 week period
  • Bleed animal
  • Can then isolate the antibodies specific to the antigen in the injection
24
Q

Explain how enzyme assays can be used diagnostically

A
  • Measure the concentrations of specific enzyme in serum -> used as a marker to assess tissue damage (e.g. ALT/AST in liver, creatine kinase following MI)
  • Measure enzyme activity by monitoring substrate/product concentrations
25
Q

Describe how you could measure the activity of an enzyme using assays

A
  • Measure the products of an enzyme controlled reaction
  • Use of fluorescent chemicals (CHEMILUMINESCENCE) or enzyme linked antibodies that change colour as more product is formed (SPECTROPHOTOMETRY)
26
Q

Explain the process of an enzyme-linked immunabsorbant assay (ELISA)

A
  • Coat well with know concentration of antigen
  • Add unknown antibodies - specific antibodies will bind to epitopes on antigen
  • Wash out mixture to remove antibodies that don’t bind
  • Add secondary enzyme linked antibody which binds to primary antibody
  • Add substrate which is converted to coloured product by enzymes linked to secondary antibodies
  • Rate of colour formation is proportional to amount of specific primary antibody
27
Q

How to radioimmunoassays differ from ELISAs?

A

Use radio-linked antibody instead of enzyme-linked

28
Q

Describe the process of WESTERN BLOTTING

A
  • SDS-PAGE transferred onto nylon and placed in solution containing primary antibodies which are specific to a particular protein epitope on the SDS-PAGE sheet
  • Primary antibodies recognise specific epitope on protein and bind
  • Secondary antibodies which are linked to a marker (fluorescent or enzyme) are specific to primary antibodies and bind thereby forming a band on SDS-PAGE where the protein of interest is located

ESA1 - Molecules, Genes And Disease (16 decks)

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