Immunotechniques Flashcards
What are some immunotechniques and what they detect?
Tools of the trade - Antibodies and types of labelling to visualise proteins
Detecting protein Immuno dot blots Western analysis Immunocytochemistry Immunoprecipitation
Detecting protein/DNA interactions in vivo - Chromatin Immunoprecipitation
Rate of synthesis - pulse chase
Describe an antibody?
2 heavy chains and 2 light chains
Light chain has a variable and constant domain - each domain is around 110 amino acids
Heavy chain has a variable and 3/4 constant domains depending on the amino acid - also around 110 amino acids for each domain
These chains are linked by di-sulphide bonds in the hinge region
Antigen binding site are made up of the variable domains of the heavy and light chains = antigen binding pocket
What are we trying to do by using antibodies?
We want to characterise the native protein
We raise an antibody by taking a peptide from the protein - around 12 aa and inject with an adjuvant
Normally in a rabbit and given a booster injection
We can isolate antibodies against the proteins of interest from the blood serum of the rabbit
The rabbit generates a library of antibodies until one antibody recognises the ‘antigen’
Describe antibody diversity?
In our lifetime we can generate 10^13 antibodies - we get this diversity from joining gene segments
These gene segments coming together is a permanent change of the DNA in the B-cells - as by rearranging the DNA we ‘kick’ out some of the DNA in between
Define monoclonal/polyclonal antibodies?
Monoclonal - a single antibody species producing a clonal cell line - hybridoma
Polyclonal - antibodies that recognise different regions and have different affinities for your protein
How do we form monoclonal antibodies?
- A mouse is injected with the epitopes of the antigen
- Isolate the spleen cells that are immune cells producing antibodies
- Fuse these cells with myeloma cells (tumour cells) to form hybridomas
To fuse the myeloma cells with the antibody producing cells we add polyethylene glycol and HGPRT (hypoxanthine guanine phosphoribosyl transferase - The hybridomas’ are screened for production of the desired antibody
- The antibody producing hybridomas are cloned in 96 well plates
- Through clonal expansion = monoclonal antibodies
Melanoma - these cell become immortalised and will keep overproducing antibodies
Describe antibody labelling?
Need to be able to visualise antibodies: Several methods have been devised to label antibodies directly or indirectly
Directly – Radioactively (125I), fluorescently using FITC (green), rhodamine (red)
Advantages: convenient, simple
Disadvantages: health risk, potentially poor signal, can be costly
Indirectly – Using a secondary antibody that recognises the constant region of the primary antibody
Secondary antibody is usually linked to a fluorophore such as FITC (green), rhodamine (red) or enzyme such as horse radish peroxidase, alkaline phosphatase - which recognises the constant region of the first antibody
Advantages: Can amplify signal – very sensitive, can use the same secondary antibody with multiple primary antibodies – cost effective
Disadvantages: have to perform 2 rounds of detection in protocol
Describe immuno dot blots?
Protein extract from 3 tissues spotted onto nitrocellulose membrane
Nitrocellulose binds proteins very strongly - and would also bind the antibody
So milk protein or BSA is used to block non-specific binding
Add antibody, incubate/wash, add second antibody, incubate/wash
Visualise antibody by testing for conjugated enzyme - either enzymatically or fluorescently
This is a quick method
We don’t know is the protein is degraded or if the antibody is cross reacted with another protein
Describe a Western blot?
Transfer SDS gel to nitrocellulose
Incubate with specific antibody
Visualise antibody
Most commonly by a secondary antibody linked to horse radish peroxidase
Location of antibody can then be determined using an enzyme assay
How do we detect proteins via Western Blotting?
Secondary antibodies are often conjugated to HRP
HRP catalyses the release of light upon the oxidation reaction with luminol and H2O2
Forming 3-aminophthalate - with electrons in the excited state, eventually emitting light
Detected with X-ray of photo detecting camera
This can be used to determine where the antibody has bound
Describe the principles used in immunoprecipitation?
We concentrate proteins if there isn’t a lot to detect
This uses proteins expressed by bacterial cells
Protein G, a cell wall protein originally isolated from Type G Streptococci
Protein A is derived from Staph A (Staphylococcus aureus)
Protein A and G bind the constant region of immunoglobulin G (IgG) with high affinity
They have preferences for which species the antibody is raised in - one is better at binding than another - could be due to the isotype
You need to look this up
Due to this binding ability, they can be used to facilitate the purification and recovery of either polyclonal or monoclonal immunoglobulins
This is because we have made the protein heavier by the attachment of the antibody
Protein A and G can be linked to agarose, magnetic beads or solid supports
Describe the workflow of immunoprecipitation?
Add an antibody to the cell lysate Add protein G agarose Spin/precipitate Remove the supernatant Wash the supernatant to remove anything non-specific Elute with SDS
The boiling at the start of SDS will separate the heavy and light chains therefore a different species needs to by used for the Western Blot
What are some uses of immunoprecipitation?
Look at proteins expressed at low levels
Post translation modifications
Identify components of protein complexes
Identify whether protein X interacts with protein Y
Describe co-immunoprecipitation?
Identification of proteins in complexes
Determining if proteins interact
- Nuclear extraction
- Add an antibody to bind to one of the 2 interacting proteins of interest
But as the protein should be interacting with the other protein it should be present in the same complex - Add the protein binding beads
- Immunoprecipitate the proteins of interest (spin them down)
- Wash anything that isn’t bound and collect the immunoprecipitated proteins
- Run SDS and Western Blot
We can then run another experiment with an antibody bound to the other protein - to see the same band on the gel
What is used to detect protein/DNA interactions in vivo?
Chromatin immunoprecipitation (ChIP) We can detect where transcription factors are binding, where modified histones are or where any DNA binding protein is
- First crosslink DNA and proteins - using formaldehyde (short link cross-linker) and isolate chromatin
- Sonicate or digest (with micrococcal nuclease) chromatin into fragments of around 500 bp
- Immunoprecipitate - use antibody against protein of interest and use protein A or G to pull it down and then wash
- Digest with proteinase K and reverse crosslinks before purifying DNA
- PCR amplify target sequences (or detect for hybridisation)
Use primers against a specific gene in the PCR reaction to determine if that region is bound by the protein of interest
