Experimental technique Flashcards
How do you make monoclonal antibodies specific for a protein?
Inject the purified protein into a rabit and then harvest the serum. Then immunoprecipitate sample to isolate the antibody - purify and concentrate it and make sure its positive to the protein.
How do you sensitize cells to an antigen?
Take RBL cells in a dish, they have IgE receptors on them. You treat them with a known antibody for an allergen e.g. DNP (anti-DNP), The mast cells will become armed with the antibody, then when you treat them with the known antigen, an allergic response will be triggered
What controls were needed to be done when researching the signalling of mast cells and how were they done?
Needed to demonstrate that impaired mast cell function was not due to impaired development of mast cells/ IgE receptor expression in Lats neg. BMMCs. They showed this by using flow cytometry to measure surface expression for IgE receptors on mast cells. Then did a western blot to show that KO of Lats in Lats neg cells was successful.
How can you look at calcium signalling in the cell?
A common method used is adding flourescent dye to the cell. It emits green light when activated with blue. Its emmision increases proportionally to the calcium levels and its restricted to the cytosol can’t go into organelles) Also used in lots of experiments is a chemical called thapsigargin. Its a calcium ATPase inhibitor which are found on the ER and replenish the ca store when its released. Blocking this means it can’t replenish the store, so it can be used to look at how claicum release from ER effects signalling. The ER also leaks a bit (we don’t know why) so ussually CaATPase counteracts this but when treated with TG it can’t.
How does a passive anaphylactic response study work?
Inject mouse with antibody for DNP (mouse Mab) - then inject mouse with DNP once antibody has been incorporated onto mast cells, and a blue dye. DNP causes an inflammatory response, part of this involves increased vasodilation and recruitment of immune cells to the tissue. When this occurs, the dye will be able to move into the tissue more the bigger the inflammatory response. Then use microscopy and quantification to measure how much blue dye is in the skin (take a clip of the ear)
What model was used to identify the Ras signalling pathway and why?
The model used was the drosophila eye because it is a very organised and ordered structure so you can see very easily if the pattern has been altered at all. The eye is made up of individual ommatidia, each made up of 22 cells, 8 of these are photosensitive (R cells). There was a mutant that lacked R7 - a missing gene was identified that codes for a protein similar to EGF (it was labelled sevenless). No sev meant that R7 fate could not be induced. They also found that if you add active Ras back into the cell you get development of the R7 neurone Was found that Bos (bridge of sevenless) on the neghbouring cell binds and activates it. This then causes binding of a couple of proteins including Sos. which in turn activates Ras. Studies in temp sensitive Sev mutants allowed the discovery of the downstream proteins in drosophila (The Sh2 containing Grb2, Sos (GEF) and Ras) - this pathway is very conserved
What makes an SDS-PAGE different to a western blot?
An SDS PAGE uses a dye and therefore identifies all the proteins on the blot. Whereas a western blot uses an antibody so identifies 1 specific protein.
What is surface biotinylation and how does it work?
A way to look at the proteins on the surface of the cell. It allows you to isolate and analyse the plasma membrane receptor you want. Cells are not permeabilised so none of the internal receptors are biotinylated. You tag the extracellular receptors using a biotin reagent e.g. Avidin and then add biotin. They have a very strong affinty for each other but are small so don’t effect the functionality of the protein. You then lyse the cell and purrify the lysate to isolate things that have biotin on them. Then you do an SDS-PAGE to isolate the receptor you want to look at
What is a problem with using SiRNAs for knockdowns?
They aren’t that reliable (lots of off target effects) so you have to use a few - if you use multiple and get the same result for each you know it is more or less accurate because it is unlikely they will all randomly do the same thing.
Explain how Annexin-V-Propidium iodine apoptosis assays work
You add Pi or 7AAD (A dye) to the cells - they are not permeable to this yet. Then you add AnnexinV-FITC which binds to the phosphatidylserine on the outer leaflet of the plasma membrane - when it binds it makes the cells more permeable to the dye. During apoptosis, the phosphatidylserine switches from the outer leaflet to the inner leaflet. So the amount of dye inside the cells is proportional to whether the cell is in early or late stage apoptosis.
What is a gene expression profile? What do the results mean?
They are an assay to show the activity of many genes at once at one point in time. Individual mRNA can be stimulated with different ligands shows that when stimulated with Interferon you get large spikes which therefore must be a target of the pathway. You can also use microarrays or RNA sequencing to get an idea of gene expression. You can then knock down different parts of the pathway and see what is no longer being expressed to get an idea of targets.
Explain how Split Gal-4 works
Involves splitting beta-galactosidase enzyme into alpha and beta parts (inactivated)- you express the different chains on single receptors, so when the receptors come together upon activation, it brings to 2 chains together and the enzyme is whole again and can change colour or can be stained with an antibody for both parts. They have to be brought together by an external force (do not have a high affinity for each other). Dimerisation is not uniform but is regulated both spatially and temporally (different tissues in different times). If not dimerised, even in the prescence of a ligand you get no signal.
How does a luciferase assay work and what does it show?
Used to determine is a protein can activate or repress the expression of a target gene. It can show a functional connection between the presence of the protein and the amount of gene product that is produced. But it can’t tell if it binds directly to DNA. Luciferase is a bioluminescent protein. The scientist produces a construct in which the regulatory region of a target gene is fused with the DNA coding sequence for luciferase (Figure 15.24). A separate DNA construct encodes the protein hypothesized to affect transcription. The scientist transfects a cell culture system, such as HEK 293T cells, with both constructs. If the protein is able to upregulate the transcription of the target gene, the cells will express luciferase. If the protein downregulates transcription, the cells will express less luciferase than normal.
What are FLAG-tags and how do they work?
A polypeptide added to a protein via recombinant DNA technology Its an artificial antigen to which specific, high-affinity mAbs have been developed. Therefore can be used to purify a protein via chromatin affinity/ immunoprecipitation, or for identifying proteins within cells. Good for if there is no antibody developed for a specific protein yet. It has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits because its mild purification procedure tends not to disrupt such complexes. It has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.
What is a co-IP assay? what is it used for?
To test if 2 proteins e.g. a receptor and mutant ligand physically interact. Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex.This works when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple members of the complex out of solution by latching onto one member with an antibody. This concept of pulling protein complexes out of solution is sometimes referred to as a “pull-down”.
