Flashcards in Lecture 17 Deck (25)
What technique is used to visualise protein localisation within a cell or tissue
What technique is used to visualise gene transcription inside cells and tissues and is quick and inexpensive
In situ hybridisation
GFP and transgenics are techniques used to visual gene expression and protein localisation, T or F
Which method is often the first port of call to visualise RNA expression due to its cheap costs and short time
In situ hybridisation
Explain how an expression plasmid is made in the process of making an antibody
The mRNA for the protein which you want to target an antibody for is extracted from a cell and converted to cDNA. This cDNA sequence is inserted into a vector containing a bacteriophage promoter. The cDNA is incorporated into the expression plasmid next to the bacteriophage promoter which is included as these promoters drive high levels of RNA synthesis and hence will produce large amounts of the protein. The promoters are also modified so that they are inducible either by chemical exposure or temperature changes to induce gene expression. These expression plasmids also contain an epitope tagging system to allow for rapid and efficient purification of the protein. These tag coding sequences are inserted in-frame and upstream of the protein of interest cDNA and are sequences to which antibodies are readily available for. These expression plasmids are then injected into bacteria which can then make the protein.
Why are expression plasmid promoter regions made to be inducible
The problem with incorporating bacteriophage promoters is, due to their high levels of expression, the bacteria in which these vectors are introduced tend to die quickly due to exhaustion. By making the promoters inducible you can minimise the time spent synthesising protein to allow the cells to survive
Explain the purpose of an epitope tagging system when creating expression plasmids in antibody synthesis
Epitope tags are short coding sequences integrated upstream of the cDNA in frame. These will be transcribed and translated with the desired protein and allow for the rapid and efficient purification of the protein. They code for peptides to which antibodies are readily available and already manufactured for.
Explain the process of antibody-affinity purification
The crude extract is poured onto a column containing beads with antibodies for the epitope tag bound to them. A pH 7 buffer is then also loaded into the column. The crude extract then runs through the column and the protein of interest is retained by binding of the antibody covered beads to the epitope tag. The rest of the crude extract travels through the column and is removed. The column then undergoes a series of subsequent washes with the pH 7 buffer until no more protein comes out of the column. The pH is then reduced to pH 3 with another buffer which breaks the interaction between the antibody and the epitope tag and protein of interest. This results in elution of the pure protein from the column.
Once the target protein, for which you want to create an antibody for, is isolated from the crude extract, how is this then used to make the specific antibodies
The purified protein is injected into an animal (i.e. rabbit, mouse, donkey) several times, typically once a month for a three-month period. After 3 months, the blood is taken from the animal and the antibodies are then purified.
What is the name of the region of a protein to which an antibody binds to
How are antibodies visualised once they are bound to a target protein
Tagging antibodies with dyes or enzymes to allow determination of where proteins are localised
What is meant by antibody sandwiches and why are they used to visualise protein localisation
Antibody sandwiches are produced by raising a secondary antibody that will bind to the first antibody. This produces an antibody sandwich of the primary antibody bound to the target epitope and then a secondary antibody bound to the first one. The secondary antibodies are usually tagged and this allows amplification of the signal. Because more than one tagged secondary antibody will bind to the primary antibody, a greater signal is produced
How are tagged secondary antibodies produced
Secondary antibodies are raised against general antibodies from the original animal species in which the primary antibodies were raised. These secondary antibodies produced in a different species will bind to any antibodies from the other species. These are then conjugated with dyes that are fluorescent allowing for the detection of protein location using specific wavelengths
Give two examples of commonly used enzyme conjugates in antibody detection
Alkaline phosphatase – turns the substrate blue. Horseradish peroxidase – turns the substrate brown
To stain a cell/tissue with a tagged antibody, the animal/cells must be chemically fixed, how is this achieved
A fixative, usually formaldehyde is introduced to cross-link proteins together
What are the two types of antibody staining and what are they used for visualising
Whole mount staining is used to visualise whole structures and tissues whereas staining on section involves antibody staining of slices creating cross section, this is often used in human samples.
Explain the process of in situ hybridisation
Start with a purified vector containing the cDNA of interest known as the template. This cDNA is incubated with RNA polymerase to make an antisense RNA probe. The antisense RNA probe will have incorporated epitope tagged nucleotides, special nucleotides with epitope tags conjugated to them. The cells are then incubated with antisense probe which will hybridise with the endogenous mRNA. Excess probe is washed off. The epitope tags often involve alkaline phosphatase which allows the mRNA to be visualised
Explain the differences seen in bicoid mRNA and protein localisation seen by using antibodies and in situ hybridisation
In situ hybridisation of bicoid mRNA will reveal its localisation in cells at the anterior region of the Drosophila embryo with defined borders. However the antibody staining for the bicoid transcription factor protein will show a different pattern. It would show a decreasing gradient of the bicoid protein, indicative of a morphogen
How is bicoid mRNA localised anteriorly in the Drosophila embryo
Contains within its 5’UTR a region that localises it at the anterior region of the cells
Explain how GFP works to provide fluorescence
GFP is excited by blue light with a wavelength of 475nm. Excitation of the GFP results in electrons within the protein increasing their energy level. The transition of these electrons back down to low energy states gives off energy in the form of light. For GFP, this energy emits is given off as green light with a wavelength of 510nm
Fluorescence always gives off an emission wavelength that is greater than the excitation wavelength and a lower energy, T or F
Explain how GFP is used in gene fusion to create transgenic lines
Firstly, you genetically engineer GFP onto the end of the last exon of the target protein by adding a GFP encoding sequence to the end of the last exon. This will be translated to a fusion protein contiaining the target protein and a fused GFP protein.
What does GFP gene fusion specifically allow that reporter constructs done
Because a functional protein of interest is produced the subcellular localization of the protein once can be studied
How does the incorporation of GFP still lead to the production of a functional protein
GFP doesn’t interact with the other protein and doesn’t impact its folding. Hence a functional protein is produced which allows for the study of where it is localised.