20.02.11 Protein based techniques Flashcards
1
Q
Name 4 protein-based techniques
A
- Western blotting
- Immunoprecipitation
- Immunohistochemistry
- Mass spectrometry
2
Q
What does western blotting do
A
-Separates proteins by electrophoresis, which is then transferred to an adsorbent membrane where antibodies detect protein of interest
3
Q
Uses of western blotting
A
- Proves the absence/ presence of protein
- Compares protein levels across different tissues/ physiological conditions/ development.
- To assess purity
- Estimate relative molecular mass
4
Q
WB step 1- Sample preparation steps.
A
- Lyse cells/ tissue to release proteins. Often buffers have protease inhibitors to prevent degradation of proteins.
- If blot is being used for quantitative purposes then protein conc should be determined (BCA assay)
- Loading dyes used to track migration of sample through gel.
5
Q
WB step 2- Protein separation by SDS-PAGE
A
- PAGE= polyacrylamide gel electrophoresis
- Proteins separated by molecular weight. Done under denaturing conditions (using SDS, a detergent).
- SDS also gives proteins a negative charge (proportional to length). Therefore proteins separated by MW not charge.
- Non-denaturing conditions needed for antibody recognition. Here samples are not heated or exposed to SDS. Proteins usually have a negative charge in alkaline buffers so migration still occurs.
- 2D PAGE. When a gradient pH is present. pH changes the charge on the protein. Proteins run to their isoelectric point (pH where net charge is zero). The next separation is then based on MW. Used to separate complex samples.
6
Q
WB step 3- Blotting
A
- Proteins transferred from gel to membrane by diffusion, an electric field or a vacuum
- Gel and paper is sandwiched between absorbent material and submerged in transfer buffer and an electrical field applied. Proteins travel towards positive charged electrode and bind to membrane.
7
Q
WB step 4- antigen detection
A
- Membrane is washed with non-fat milk to prevent non-specific binding of antibody.
- Direct detection- primary antibody detects antigen on blot and is labelled
- Indirect detection. Primary antibody binds to antigen and a secondary labelled antibody binds to the primary.
8
Q
WB step 5- visualisation
A
- chomogenic= enzyme coupled antibodies, e.g. HRP (horseradish peroxidase). Substrates added which yield visible coloured products
- Chemiluminescent detection. Substrates produce a chemiluminescent product (CPD), which is detectable on X-ray film
- Fluorescent detection- secondary antibodies are coupled to fluorochromes.
9
Q
What is immunoprecipitation
A
- Antibody immobilized support that enriches/ purifies a protein from a sample.
- Purified proteins can then be quantified/ analysed by other techniques e.g. Western blotting
10
Q
Two methods for immunoprecipitation
A
- Pre-immobilised approach. Antibody is pre-immobilised onto an insoluble support and incubated with lysate containing the target protein. Protein binds to antibody, remaining material washed away. The protein of interest is then eluted.
- Free-antibody approach. Free unbound antibody forms immune complexes in lysate. Complexes are then isolated using beads. Beneficial if antibody has weak affinity for antigen or binding kinetics are slow.
11
Q
What is co-immunoprecipitation
A
- Co-IP can identify physiologically relevant protein-protein interactions.
- Protein specific antibodies indirectly capture proteins that bind to the target protein.
- Uses= identify new binding partners, binding affinities, biding kinetics, functions of target proteins.
- Pull down assays= not using antibodies, i.e. covalent attachment or affinity tags
12
Q
What is Chromatin IP
A
- Selective enrichment of a chromatin fraction containing a specific protein.
- Antibodies used to immunoprecipitated proteins of interest along with associated DNA.
- Uses= gives insight into gene regulation
13
Q
Application of IP in routine testing
A
- Useful to determine effect of missense mutations on protein-protein interactions, half-life, subcellular location.
- Not really amenable to routine testing.
14
Q
What is immunohistochemistry (IHC)
A
- Localises specific antigens in tissues using antibodies. Typically visualised using an antibody conjugated with an enzyme (peroxidase) which catalyses a colour-producing reaction.
- Gives evidence on presence/ absence/ localisation of a protein. Also tissue structure and cellularity.
15
Q
Difference between immunohistochemistry (IHC) and immunocytochemistry (ICC)
A
- IHC= uses sections of tissue (cells surrounded by matrix)
- ICC= uses cells with matrix removed, eg. cultured cells/ suspension/ smear.
