Non Genetic Analysis Flashcards
What can antibodies help us do
Find where a protein is in a cell.
Find where a protein is in an organism
What can RNA in situ help us find
Where is the gene transcribed
What can GFP help us to do
Visualise gene expression and protein localisation in living cells.
The first step of making antibodies:
Why is a promoter needed and why does it need to be inducible
How is the promoter turned on
A bacterial plasmid has the cDNA of interest inserted into it.
The plasmid also contains a bacteriophage promoter which will cause the inserted cDNA to be over expressed and so vast amounts of the protein is produced.
The promoter has to be inducible or the bacteria would die due to loss of energy because all of the bacteria’s energy will be devoted to making the protein.
The promoter is silent to begin with until the bacteria have replicated many times. Then they are all turned on and lots of protein is made.
The promoters are induced by raising the temperature or adding chemicals.
Second step of antibody production:
How is crude extract made
After the induction a few hours will pass before the bacteria start to die.
The bacteria are centrifuged into a pellet and are lysed by adding detergent to break down the cell membrane and open up the cells to make the crude extract.
How can the protein be purified from the crude extract
Epitope tagging is very rapid and easily available.
The tag is fused to the cDNA during the cloning. And when the protein is made it will have the tag too so it can be found.
This is antibody affinity purification.
The crude extract is poured into a column.
This has beads which antibodies on then which are complementary to the tag.
The protein of interest will stick to the beads. Everything else is washed out.
The column is rinsed and eluted with a pH3 buffer to break the bonds. And the protein can be collected at the bottom.
What can you do now you have the purified antibody.
Inject into a rabbit several times over three months and the rabbit will have an immune response.
The rabbit will make antibodies for the injected protein and these can be extracted and purified.
What is an epitope
The antibody will bind to small regions of proteins with very high specificity called an epitope.
How can antibodies be labelled
Which is more sensitive
What about using two antibodies
Some can be fluorescently dyed so we can detect the location using a specific wavelength of light.
Or we can use enzymes such as alkaline phosphatase whose substrate turns blue.
Or horseradish peroxidase whose substrate turns brown. Using enzymes is better because it is more sensitive.
Or you can use two antibodies. The 1 prime binds to the protein and the 2 prime bind to the 1 prime and carry the tag.
The 1 prime will be made in the rabbit.
What needs to happen when we add the antibody to the tissue.
The tissue has to be chemically fixed using formaldehyde to stabilise the structure
So the proteins will stay where they are and the tissue won’t rot.
Then the tissue and antibody are incubated together so they bind.
Excess antibody is washed off.
How do we make the RNA probe for in situ hybridisation
How do we tag the probe
The cDNA of interest will be in a plasmid.
An in vitro synthesis of RNA is done from the cDNA in the plasmid to make the RNA probe.
The nucleotides used to make the RNA are ectopically tagged.
Or we can tag the probe using DIG label.
An antibody will bind to it and alkaline phosphatase is blue.
What do we do after we have the probe for in situ hybridisation
Incubate the chemically fixed tissue with the probe and allow them to hybridise.
Wash off excess probe.
Detect where the probe has bound to see where the gene is expressed.
What wavelength is GFP excited by
What wavelength will it emit
Why does this happen
475nM.
510nM green light.
The protein absorbs the higher energy light and one of the electrons is shifted out of the normal orbit.
This is temporary and when it returns to its normal position it emits the lower energy light.
Generating a GFP transgenic line
What are the two ways of doing this and compare them
A plasmid will contain the genomic region of interest.
Gene fusion - Genetically engineer GFP onto the end of the last exon
Reporter construct - remove all the exons except for GFP so the gene is replaced.
Gene fusion is more complicated and can follow the sub cellular localisation of the protein. will not affect the function of the protein and can be done on live animals
Reporter construct shows where in the animal it is expressed. You need to know the regulatory sequences for it to work.
What do you do after you have the GFP fusion gene
Integrate it back into the genome.
Microinject a solution of the DNA into the one cell zygote and the DNA will then randomly insert into the genome.
DNA repair enzymes in the host allow for the added gene to integrate into the host genome.
If this is done in nice it would be inserted into ES cells.
