Lecture 1 Flashcards
What type of technique is western blotting?
a protein detection technique
What are the 3 main stages of western blot?
SDS polyacrylamide gel electrophoresis (SDS-page) - separates protein in a gel according to size.
Protein transfer onto a high-affinity protein-binding matrix
Sequential incubations with primary and secondary antibodies to detect target protein (the western blot)
Describe SDS-PAGE.
Protein mixtures (cell lysates) are denatured in SDS solution by boiling. The secondary and tertiary structures become denatured so protein goes to its core sequence.
SDS gives each protein an overall negative charge.
Polyacrylamide gel is made that resolves the proteins according to size - proteins migrate towards a positive electrode (negative electrode at the bottom so proteins move upwards based on MW).
Describe protein transfer in western blotting.
Proteins on the gel are electro-transferred to a nitrocellulose membrane.
MW protein markers transfer onto the membrane indicating the transfer has worked.
Describe the western blot stage 1 (membrane blocking).
Nitrocellulose membrane has a high affinity for protein so milk proteins non-specifically bind and block aberrant antibody binding in subsequent steps.
Describe the western blot stage 2 (primary antibody incubation).
Apply a primary antibody targeted to the protein of interest.
Primary antibody binds specifically to the epitope region of the protein.
Describe the western blot stage 3 (secondary antibody incubation).
Secondary antibody detects the primary antibody.
Secondary antibody must be anti whatever the primary antibody was so it can bind to the primary (example if primary Ab is mouse then secondary Ab should be rabbit anti-mouse).
Enzyme conjugate (horse-radish peroxidase) on Fc fragment is important for physical detection.
Describe western blot stage 4 (detection of bound antibody complex).
Activate the HRP enzyme when a substrate is added that emits light.
Can be detected using a digital image - detects photons.
Why is a controller required for western analysis?
Gives an idea of the loading on the gel and how well it was equally loaded between each cell.
What is a suitable control for western blot analysis?
Using proteins such as alpha-tubulin and beta-actin as these are expressed at equal levels in cells,
If the bands are equal then any differences observed in the protein of interest are to do with manipulation of the cell NOT loading different amounts.
What is another suitable control for western analysis?
Protein concentrations of each sample can be determined using a spectrophotometer prior to loading on a gel.
Once each sample concentration is known, equal quantities of protein can be loaded onto a gel.
What does ChIP stand for?1.
Chromatin immunoprecipitation
What are the 3 basic principles of ChIP?
- ChIP is an informative technique that allows analysis of protein function and epigenetic changes during the process of transcription.
- It can be applied to study other cellular events that requires chromatin processing such as DNA replication and repair.
- Relies on the enrichment of a target protein using an antibody in a process called immunoprecipitation.
What is the enzyme that transcribes protein-coding genes within DNA into RNA?
RNA pol II
What are RNA pol I and III involved in?
Transcribing ribosomal (pol I) and tRNAs (pol III) for the subsequent process of translation.
What do transcription factors do?
Facilitate the loading of RNA pol onto target gene promoter elements allowing for transcription to take place.
What are the 5 steps of ChIP?
- cross-link protein and DNA with formaldehyde
- lyse cells and sonicate to fragment chromatin
- add antibody bound beads to bind protein DNA complexes (immunoprecipitation)
- reverse cross-links between protein-DNA and proteinase K treat samples to remove protein from mixture
- purify DNA and set up quantitative PCR or DNA sequencing
Describe cross-linking protein DNA and protein-protein interactions in ChIP.
Formaldehyde introduces covalent cross-links between DNA and protein that sticks them together.
For transcription factors, cross-linking them to DNA is important as their interaction with promoter elements is transient and may not be detected otherwise. Gluing them to DNA improves the efficiency you can detect DNA interactions.
Describe cell-lysis and chromatin fragmentation by sonication in ChIP.
Formaldehyde treated cells lysed using buffers to enrich for nuclear proteins.
Samples sonicated (high frequency sonic waves applied) to break phosphdiester bonds within DNA (ideal size is between 200-500 base pairs).
Process results in fragmented chromatin that retains the cross-linked DNA-protein interactions.
Describe immunoprecipitation in ChIP.
Antibodies targeted to a specific protein or a control IgG are mixed with magnetic beads and added to the mixture.
Control IgG controls for non-specific protein DNA interactions.
Antibody binds to the target protein complexed to DNA and enriches the protein-DNA complex using magnetic-based separation,.
Describe why using control IgG antibodies is important in ChIP.
Confirms specific protein binding to the target genomic loci.
Some antibodies have the capacity to interact non-specifically with proteins making ChIP experiments difficult to interpret and can cause false positive data.
Using control IgG (which has no specific affinity for any cellular protein) allows for distinction between specific antibody-protein interactions and non-specific protein immunoprecipitation.
Describe cross-link reversal and proteinase K treatment for ChIP.
After immunoprecipitation.
Cross-link reversal to breakdown interaction between receptor and DNA.
Proteinase K degrades the protein as we need the DNA to be left.
Describe DNA purification and PCR analysis of ChIP.
Purify the immunoprecipitated DNA and then perform a PCR to see if DNA sequences of interest have been pulled out.
Primer specific to the gene to see if the receptor has bound to the promoter element of the gene.
Compare the enrichment of the receptor to the control IgG.
What does ChIP linked with total DNA sequencing provide?
A snapshot of global transcription factor/histone modification enrichment across the genome an is useful for understanding how transcription factors control cell fates.
What is a mole of a substance equal too?
Molecular weight in grams
What is molarity?
A measure of concentration of a solution.
How would you describe a 1 molar solution?
The molecular weight of the substance in grams dissolved in a litre of water.
Eg. Glycine MW = 74 so 1M glycine solution = 74g in a litre of H2O
How would you calculate the dilution factor?
Initial concentration divided by final concentration.
