Antibodies and Immunodetection Flashcards
(33 cards)
What methods in imumunodetection can be used to detect protein?
Immuno dot blots, western analysis, immunocytochemistry and immunoprecipitation.
What method can be used in vivo to detect protein/DNA interactions in vivo?
Chromatin precipitation
What method can be used to detect the rate of synthesis?
Pulse chase
What method can be used to measure synthesis in vitro?
Wheat germ extract and GST pull down experiments
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal are a population of antibodies that recognise different regions and have different affinities for your protein whereas monoclonal is a single antibody species produced using a clonal cell line called a hybridoma.
How are monoclonal antibodies produced?
Immunisation of a mouse using an antigen, the immune cells are then isolated as antibody-forming cells and fused with tumour cells to form a hybridoma. The hybridoma is screened for production of desired antibody and cloned. Monoclonal antibodies are produced through clonal expansion.
What methods are their to directly label antibodies?
Radioactively or fluorescently
What are the advantages of directly labelling antibodies?
Convenient and simple
What are the disadvantages of directly labelling antibodies?
Health risk, potentially poor signal and can be costly.
What methods are their to indirectly label antibodies?
Use a secondary antibody that recognises the constant region of a primary antibody, the secondary anybody is usually linked to a fluorophor such as FITC (green), rhodamine (red) or enzyme.
What enzymes can be linked to the secondary antibody in indirect labelling?
Horseradish peroxidase and alkaline phosphatase.
What are the advantages of indirectly labelling antibodies?
Can amplify a signal, therefore very sensitive, can use the same secondary antibody with multiple primary antibodies, therefore cost effective.
What are the disadvantages of indirectly labelling antibodies?
Have to perform two rounds of detection in protocol.
How are immuno dot blots carried out?
Protein extract is spotted on a nitrocellulose membrane and it is analysed using an antibody.
Describe the steps in performing a western blot?
1) Extract protein form cells/tissues and carry out electrophoresis- separation based on size.
2) Transfer to nitrocellulose membrane
3) Calculate Mr from distance moved
4) Incubate with specific antibody, wash off
5) Visualise where the antibody was using, most commonly, secondary antibody.
What is used in SDS PAGE and why?
Negatively charged SDS to keep the protein denatured and eliminate the intrinsic charge to allow the protein to migrate through the gel solely by charge. Add beta-mercaptoethanol or dithiothreitol to keep the protein denatured and remove disulphides bridges between cysteine residues.
How is the nitrocellulose membrane first prepared?
It is blocked, to prevent non-specific binding to the membrane, by any cheap protein eg. Milk marvel powder
How accurate is it calculating Mr from migration through and SDS PAGE?
Glycosylated proteins show larger Mr due to bulkiness but otherwise accurate.
What is the most popular method of detection using secondary antibody?
Horse radish peroxidase.
How does horse radish peroxidase work?
Catalyses the release of light upon the reaction with luminol/H2O2 and can be used to determine where the antibody is bound.
What are protein A and G?
Protein G is a cell wall protein isolated from Type G Streptococci and protein A is derived from Staph A. Both bind in the constant region of IgG with high affinity and can be used to facilitate the purification and recover of either poly or monoclonal antibodies.
What can protein A and G be linked to?
Agarose, magnetic beads and solid supports.
Describe the steps of immunoprecipitation?
1) Add antibody to cell lysate
2) Add protein G agarose
3) Spin to precipitate
4) Remove supernatant
5) Elute with SDS
What are the uses of immunoprecipitation?
To look at proteins expressed at low levels, post translational modifications, to identify components of protein complexes and to identify whether protein interact.
