TF part2 Flashcards
What is the purpose of experiments that show binding of a factor to DNA?
To identify whether a transcription factor interacts with DNA.
What two things can ChIP be used to determine?
<p>Whether a TF binds to a gene, and where in the gene it binds.</p>
<p>This interaction can be analysed for just a single gene or for multiple gene targets of a transcription factor.</p>
<p>Steps of Chip</p>
<p>Step 1. All proteins that are interacting with DNA are crosslinked.</p>
<p>Step 2. The chromatin (DNA) is isolated then fragmented into short pieces of 100bp - 1000bp.</p>
<p>Step 3. An antibody that has an epitope that binds an antigen on the transcription factor is added.</p>
<p>Step 4. Agarose beads are added, which bind to the antibody. This is done to pull down the protein DNA complex during centrifugation</p>
<p>Step 5. The DNA - protein crosslinks are reversed and the DNA purified to be used in downstream applications.</p>
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What is the size range to which chromatin is fragmented in ChIP?
100bp - 1000bp.
What is the purpose of sonication in ChIP?
To break DNA into smaller fragments.
What is done with the DNA-protein crosslinks in step 5 of ChIP?
Reversed, and the DNA is purified.
In ChIP, what should primers be designed to bind to?
<p>TF binding sites- Researchers will only amplify DNA regions associated with TF of interest</p>
What does the pellet after centrifugation contain in ChIP?
Agarose beads with transcription factors.
What does a positive control tell you in ChIP-PCR?
Immunoprecipitation and PCR went correctly.
What is the purpose of PCR on genomic DNA from ChIP?
<p>To determine if and where a TF binds to a gene promoter- After isolating sample from centrifugation</p>
For multi-gene analysis, what method can be used to investigate all gene targets of a TF?
ChIP-seq.
What is done with the DNA sequences after immunoprecipitation in ChIP-seq?
Sequenced using NGS.
Electrophoretic Mobility Shift Assay (EMSA)
A method to show binding of a transcription factor to DNA.
What does EMSA tell you?
Which of these regions the TF actually binds to.
Electrophoretic Mobility Shift Assay
<p>Can determine if a transcription factor binds to a DNA piece<br></br>To investigate whether a specific protein interacts with a specific fragment of DNA..</p>
What is needed for EMSA?
<p>Purified transcription factor or the binding domain of the transciption factor and a DNA fragment that can be made by synthesis or PCR.</p>
What are other names for EMSA?
Gel shift assay or gel retardation assay.
Gel shift assay
Another name for Electrophoretic Mobility Shift Assay (EMSA).
Gel retardation assay
Another name for Electrophoretic Mobility Shift Assay (EMSA).
What is used in EMSA instead of live cells?
A manufactured DNA piece with a predicted binding site.
What is used to artificially express protein or binding domain in EMSA?
A cloning vector inserted into bacteria.
How are TF and DNA separated in EMSA?
Using agarose or polyacrylamide.
How does TF binding affect movement through the gel?
Makes movement slower.
<p>Why can't you use Chip for determinign sequences close together</p>
<p>There is a limit to the size of the fragments cleaved as well as how large primers as well as the space between primers can be</p>
What are the steps of the Electrophoretic Mobility Shift Assay?
Step 1. A DNA fragment is synthesised (often incorporating a label).
Step 2. The DNA is mixed with protein/s. The transcription factor will then bind to its sequence in the DNA fragment.
Step 3. Agarose or polyacrylamide gel electrophoresis is used to resolve the DNA fragment.
Step 4. The DNA bands on the gel are visualised in a number of ways.
By incorporating a ³²P - labelled dNTP or using DIG.
- DIG is a hapten that can be subsequently detected using anti - DIG antibodies. - If a lot of DNA is used in the assay, then the DNA can be stained using ethidium bromide or Sybr green at electrophoresis
Circular DNA vector that contains a reporter gene.
Also contain: - a multiple cloning site (MCS) - antibiotic selection marker.
Immunocytochemistry
How does RT-PCR and qPCR dtermine transcription levels
RT - PCR and qPCR amplify cDNA that has been made from mRNA transcript. As such, the presence of transcript (from the target gene) can be determined using primers specific to the transcript. RT - PCR can only indicate the presence of the transcript, whilst qPCR can quantify transcript levels.
Western blotting steps
Step 1. Extract total cellular proteins. Step 2. Perform SDS - PAGE to separate all cellular proteins based on size. Step 3. Transfer proteins to a membrane such as nitrocellulose. Step 4. Block the membrane (with non - fat milk or BSA) to reduce non - specific binding of antibodies to membrane. Step 5. Incubate with primary antibody that targets protein of interest then wash membrane thoroughly to remove excess and non - specifically bound antibody. Step 6. Incubate with secondary antibody that targets the primary antibody. The secondary has a label that allows for detection. Wash membrane thoroughly to remove excess and non - specifically bound antibody. Step 7. Detect the label, for HRP this requires exposure of membrane to chemiluminescent reagent, which will emit light in the presence of HRP.
Reporter gene steps
-Step 1. Clone target gene promoter containing transcription factor binding site into the reporter vector. Cloning of the gene promoter can be done by PCR. By adding restriction sites to the end, these can be used to insert the DNA into complementary sites within the MCS.
-Step 2. The vector is transformed into competent E.coli cells then plated onto LB agar plates containing ampicillin. Only E.coli expressing the ampicillin resistance gene from the vector will form colonies. An individual colony is then grown in ampicillin - containing LB broth to amplify the vector, which is then isolated.
-Step 3. The isolated vector is then transfected into cells. A cell line such as the HEK293 cell line is often used as it has a high transfection efficiency. In the presence of a stimulus that activates the transcription factor of interest, the protein will bind to the binding site in the cloned promoter thereby causing expression of the luciferase gene.
Step 4. The HEK293 cells are lysed and the enzyme substrates added, as in the example below. The light emitted can be detected on a luminometer.
Methods in Gene expression analysis
-Looking for changes in transcript levels of the transcription factor gene target:
-Quantifying changes in protein levels of transcription factor and protein of target gene:
-Observing localisation of proteins:
Looking for changes in transcript levels of the transcription factor gene target:
Reverse Transcriptase - PCR • qPCR
RT-PCR steps
Step 1. Extract total RNA or Extract mRNA from cells. Step 2. Synthesise cDNA from the mRNA. Step 3. Use target specific primers to amplify region of interest by PCR. Step 4. View PCR products on an agarose gel for RT - PCR. Quantify fluorescence levels in qPCR.
What is immunocytochemsitry used for together with microscopy
To observe localisation of proteins within a cell
-it uses fluorescent antibodies
Observing localisation of proteins steps
Step 1. Cells are permeabilised to expose antigens on protein. Step 2. Non - specific sites are blocked. Step 3. Incubate with primary antibody that target s protein of interest. Step 4. Incubate with secondary antibody that target s the primary antibody. The secondary has a label such as a fluorescent label. Step 5. Fluorescence microscopy allows for detection and visualisation of fluorescence in cells.
