L8 The uses of antibodies in the study of cell signalling

Question 1

What are six main uses of antibodies in research/therapy?

Answer 1

Western blotting, ELISA, IHC, ICC, IF, IP

Question 2

Besides the main six uses of antibodies in research and clincially, what are three other uses for them?

Answer 2

Purification of proteins, exploring protein/protein interactions (e.g signalling complexes), in-vitro kinase assays (radio & non-radioactive)

Question 3

What are the two steps of western blotting?

Answer 3

Electotransfer (via SDS-PAGE or some variaton on this assay)

Antibody detection

Question 4

What are the three typical western blotting reporters?

Answer 4

Colorimetric, chemiluminescence, fluorescence.

Question 5

Briefly how do colorimetric, chemiluminescence and fulorescence western blot assays reporters differ visually?

Answer 5

Colorimetric - positivie result is conveyed by presence of colour visible to human eye.

Chemiluminescence - positive band visible using detection machinery e.g. X-ray.

Fluorescence - with antibody linked to fluorophore that releases specific wavelength/colour light, multiple can be used to visualise and test for multiple results.

Question 6

Briefly describe the principles of indirect ELISA

Answer 6

Used to detect antibody in sample.

Immobilised antigen used. Primary antibody (investigated antibody) ran over solid phase, washed. Then conjugated secondary antibody (specific to primary antibody Fc region) ran over. Wash.

The now immobilised conjugate on secondary antibody reports on presence/absence of primary antibody against antigen.

Question 7

What are the principles that immunoprecipitation works upon?

How is immunoprecipitation performed?

Answer 7

Used to purify protein of interest from cell lysate.

Antibody added against POI, binds.

Protein A or G added to make antibody-protein complex insoluble. These proteins bind to antibodies and take them out of solution.

Centrifuge, pellet forms at bottom which is this complex. Remove supernatant and wash pellet.

Analyse pellet via SDS-PAGE.

Question 8

In immunoprecipitation, what steps can be taken to avoid centrifugation?

Answer 8

Can be performed with magnetic beads linked antibodies.

Question 9

What would you consider when deciding to use either protein A or G in IP?

Answer 9

Different species’ of animals antibodies have unique binding profiles for A and G. e.g. Rat IgG2a have very strong Protein G binding but no protein A binding.

Guinea pig IgG1 has very strong Protein A binding, but weaker protein G.

Question 10

What are the two main receptor signalling pathways used in cell signalling?

What do they recognise?

Answer 10

GCPR’s - hormones, neurotransmitters, peptides, proteins, calcium ions, protein kinase A/C/G and Tyr-kinase receptors. (GF receptors) - insulin, egf, PDGF

Question 11

PTM’s are very important in protein function. What are the general PTMs?

Answer 11

Glycosylation, methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, isoprenylation, proteolysis.

Question 12

What role do PTMs have on p53?

Answer 12

Vastly alter p53’s structure and thereby function.

Tumour suppressor often mis-regulated in cancer. PTMs can change function from tumour-suppressor to oncogene!

Question 13

Kinases perform what PTM (most common PTM)

Answer 13

Phosphorylation

Question 14

Is protein phosphorylation reversible?

Answer 14

Yes reversible process.

Question 15

What are the three amino acids residues which are phosphorylated?

Answer 15

Serine/threonine kinases (e.g. PKA, PKC)

Tyrosine kinases (e.g. TKR (EGF) and non-receptor TK, Src and JAK).

Question 16

What techniques can be used to determined whether protein of interest is phosphorylated?

Answer 16

Immunoprecipitation and SDS-PAGE, directed by radiolabelling.

Label cells with [³²P]-orthophosphate. After SDS-PAGE, gel can be dried onto film and visualised using autoradiography.

Question 17

How can it be investigated which types of amino acids are phosphorylated?

Answer 17

Phosphoamino Acid analysis.

Extract protein from gel, hydrolyse. Analyse using 2D thin-layer chromatography (TLC) or HPLC.

Determines which types are phosphorylated e.g. Tyrosine, serine or threonine.

Question 18

How can the site of phosphorylation be determined?

Answer 18

Two-dimensional phosphopeptide mapping of tryptic digest.

HPLC of trypic digest.

Site-directed mutagenesis

2D Gel EF

Question 19

In phosphopeptide mapping, how does the sample run through the gel?

Answer 19

First dimension electrophoresis (on charge or phosphate number) Second dimension TLC on size.

Distinct spots seen indicating amount of phosphorlyations.

Question 20

How can site-directed mutagenesis be used to investigate phosphorylation sites?

Answer 20

Mutating different residues on a protein, then analysing sample for presence or absence of phoshphorylation against native regular PTM conditions.

e.g. if by mutating Thr³⁰⁷ phosphorylation isn’t observed in protein when it is in native, then good indication that this is the site.

Question 21

Can 2D-PAGE be used to study PTM?

Answer 21

Yes, first gel isoelectrically focuses proteins of interest. Phosphate group conveys charge.

Question 22

How can image analysis be used on a 2D-PAGE gel to identify and isolate POI?

Answer 22

POI has known isoelectric charge and weight.

On complex gel, imaging software can identify this on x/y axis of gel and point out which spot to excise for further analysis.

Question 23

Briefly describe how antibodies can be used to study protein phosphorylation?

Answer 23

By use of antibodies raised to target phosphorylated-Tyr/Ser/Thr. (Anti-pTyr/anti-pSer/anti-pThr).

Question 24

As examples, what are three uses of anti-phosophotyrosine antibodies?

Answer 24

Looking for gross changes in protein tyrosine phos.

Minotiring tyrosine phos. of specific proteins.

Immunoprecipitation of tyrosine phosphorylated proteins.

Question 25

How can gross changes in protein tyrosine phos. be identified?

Answer 25

Western blotting, IHC, ICC.

Question 26

How are phospho-specific antibodies generated?

(clue, involves hapten carrying)

Answer 26

Rabbits/mice immunised with synthetic 12-14aa peptide containing the phospho-amino acid (coupled to KLH, carrier protein to this hapten).

Antibodies then purified using protein A / peptide affinity chromatography.