SFP20: Proteins As Research Tools Flashcards
What is an antibody?
Protein that recognises and bind to antigens
What is an antigen?
A protein on a foreign object that stimulates the immune system to produce antibodies
On an SDS page of an antigen… what would you see?
Two heavy and two light chains
The two chains of each are the same so you would get one band at 55kDa and one band at 25kDa
characteristics of polyclonal antibodies
- cheap to produce
- mixed population of antibodies
- may bind to different areas of target molecule
- tolerant of small changes in protein structure
Characteristics of monoclonal antibodies
- expensive to produce
- single antibody specific
- will only bind single specific site
- infinitely renewable
- may only recognise a particular protein form
Describe polyclonal antibody production
1) inject antigen into rabbit
2) antigen activates B cells
3) plasma B cells produce polyclonal antibodies
4) obtain antiserum from rabbit containing polyclonal antibodies
What does staining with coomassie allow?
Each band can represent one or more proteins at that particular weight
What does western blotting allow?
It uses antibodies to detect individual proteins within the gel
How can missense/ mutant proteins now be detected?
Use mutant-specific antibodies… have wild-type or mutant peptide
What is ELISA?
Enzyme-linked immunosorbent assay (ELISA)
- very similar principle to dot-blot
- sample simply binds to wells of microtitre plate
- Ab selectively recognise… rest is like a western plot
- directly measure absorbance of substrate in wells
What is immunohistochemistry?
- detecting proteins in (fixed) cells
- localising proteins (endogenous antigens) in thin tissue sections on glass slides
- visualise by light microscopy - spatial information
How is immunofluorescence different to immunohistochemistry?
Rather than using enzyme linked secondary antibodies, you can use fluorescent tagged either primary or secondary
What is TMA (transcription mediated amplification)?
- a type of immunohistochemistry
- screen multiple (small) samples in parallel for the same antigen
- needle biopsies applied to the same glass slide
- 13/80 tumours stained positively for p53 - analyse multiple samples simultaneously
- caveat- heterogeneity of tissue sampled
What is immunoblot electron microscopy?
- similar to IHC/IF but gold conjugated secondary antibodies and detection by EM
- different primary/secondary antibodies combinations with different size gold particles allow detection of to different proteins (co-localisation)
- gold particles are electron dense
- extremely high resolution, detection of endogenous proteins
What is Immunoprecipitation?
- antibodies can be used to selectively capture (purify) other proteins from complex mixtures e.g. cell extracts
- actually a form of affinity chromatography
