L3a- More Experiments Eg Elisa/immunofluorescence Flashcards
Give 2 ways you could study genes involved during process of differentiation through growth factors
Candidate approach- literature search for genes you think are important and design primers, amplify and compare differentiated vs non differentiated via qpcr - limited info
Global analysis - rna sequencing of 2 cell populations to see expression / differential gene expression of particular genes before and after eg differentiation
Explain steps of rna sequencing which you would do to compare transcriptomes
Extract rna from cells
Fragment
Convert to cDNA through RT
Ligate adaptors and amplify
NGS sequencing
Align reads with the genome / transcriptome
Determine levels of transcript via = gene expression upreg or downregulate between cells
When scientists did this looking at before gf treatment vs cells treated found differential gene exp patterns of synaptophysin. (Based on RTqpcr to validate the rna seq data) do this mean there was more of the protein present? How would you validate this in 4 ways
No
Need to use western blot (quantify the conc present), immunofluorescence, flow cytometry (quantify protein by ab) or Elisa to determine if protein present and quantify
How does indirect immunofluorescence work
Add specific ab to your protein
Then secondary ab with conjugated fluorophore which emits light at specific wavelengths
Detect in fluorescence microscope where your protein is in cell
How would you stain nucleus in immunofluorescence
Intercalating dye
How can you get multi colour imaging
Use different fluorophores so emit at diff wavelengths
What is the main ad of indirect over direct
No manipulation of protein so get wild type localisation info
What are the issues with indirect
Takes long time and expensive ab
Also ab may not be available for the protein of interest
How does direct work whcih negates need for ab
Fusion protein of gfp-synaptophysin produced inside an expression vector
Transfect cells with the plasmid
During translation gfp will emit light at specific wavelength for detection
What are the disadvantages here
Fusion protein may affect the proteins activity / location
Some cells cannot be transfected
Which 2 ways could you make sure that this light is showing synaptophysin and that ab aren’t binding something else
1- could perform experiment in cells lacking synaptophysin
2- could block primary ab binding to protein epitope
Both would result in no light
How is Elisa different in the way you can quantify protein
Specifically quantifies using secondary ab which is conjugated with an enzyme causing colour change not chemilinescent
What is sandwich Elisa for protein quantification in cell lysate
Capture ab attached to well and then lysate added first then primary and secondary
What is required to be made to determine conc from spectrophotometer od readings
A standard curve of known conc
How many replicates would you do on the micro tire plate
3 for each conc and 3 for each sample eg treated and non treated
