Molecular Techniques & Diagnosis of Proteins Flashcards
What is serum protein electrophoresis?
Test that examines specific serum proteins in blood called GLOBULINS
How many major bands would you expect in the gel for serum protein electrophoresis? What do they represent?
5 major bands representing the globulins in the blood:
- ALBUMIN
- α-1-globulin
- α-2-globulin
- β-globulin
- γ-globulin
How would an abundance of a serum protein be identified on the gel?
DARKER BAND
What stain is normally used in serum protein electrophoresis?
- PONCEAU S stain
- Stains bands BLUE
Explain how protein gel electrophoresis allows separation on the basis of size AND charge
- 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
What does the speed of travel of a protein through gel depend on?
- 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
Explain the clinical significance of serum protein electrophoresis
- 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
What method could you use to separate proteins purely on the basis of SIZE?
SDS-PAGE
Why does SDS-PAGE use unfolded proteins?
- Need proteins to have a singular charge
- Folded proteins have an intrinsic charge
Explain the action of SDS and β-ME in SDS-PAGE
- 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
How does SDS-PAGE allow us to identify the presence of proteins?
- 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
Explain how ISOELECTRIC FOCUSING can be used to separate proteins purely on the basis of CHARGE
- 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
What is 2D-PAGE used for?
- Allows the separation of complex protein mixtures
- Important for diagnosing disease states in different tissues
Explain the process of 2D-
PAGE in the identification of proteins
- 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)
Briefly explain how proteins can be identified using PROTEOMICS
- 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
What is the difference between proteomics and molecular diagnosis?
- PROTEOMICS is the analysis of all proteins expressed in a genome
- MOLECULAR DIAGNOSIS is the analysis of a single purified protein
Give 2 examples of enzymes which can perform specific proteolytic cleavage
- 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
What is an EPITOPE?
- Few amino acids present on an antigen
- Antibodies can recognise specific epitomes on antigens and bind to them complementary
What are POLYCLONAL antibodies?
- Multiple different antibodies that are specific to 1 antigen which contains multiple epitopes
- Produced by many B lymphocytes
What are MONOCLONAL antibodies?
- Identical antibodies which are specific to 1 antigen which contains 1 epitope
- Produced from 1 B lymphocyte
Explain how you would isolate monoclonal antibodies
- 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
What is WESTERN BLOTTING used for?
- Detection of proteins on SDS-PAGE gel using antibodies
- Can detect specific proteins present in mixtures
Explain how polyclonal antibodies can be isolated
- 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
Explain how enzyme assays can be used diagnostically
- 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
Describe how you could measure the activity of an enzyme using assays
- 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)
Explain the process of an enzyme-linked immunabsorbant assay (ELISA)
- 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
How to radioimmunoassays differ from ELISAs?
Use radio-linked antibody instead of enzyme-linked
Describe the process of WESTERN BLOTTING
- 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
