L3a- More Experiments Eg Elisa/immunofluorescence Flashcards

1
Q

Give 2 ways you could study genes involved during process of differentiation through growth factors

A

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

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2
Q

Explain steps of rna sequencing which you would do to compare transcriptomes

A

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

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3
Q

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

A

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

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4
Q

How does indirect immunofluorescence work

A

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

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5
Q

How would you stain nucleus in immunofluorescence

A

Intercalating dye

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6
Q

How can you get multi colour imaging

A

Use different fluorophores so emit at diff wavelengths

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7
Q

What is the main ad of indirect over direct

A

No manipulation of protein so get wild type localisation info

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8
Q

What are the issues with indirect

A

Takes long time and expensive ab

Also ab may not be available for the protein of interest

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9
Q

How does direct work whcih negates need for ab

A

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

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10
Q

What are the disadvantages here

A

Fusion protein may affect the proteins activity / location

Some cells cannot be transfected

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11
Q

Which 2 ways could you make sure that this light is showing synaptophysin and that ab aren’t binding something else

A

1- could perform experiment in cells lacking synaptophysin
2- could block primary ab binding to protein epitope

Both would result in no light

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12
Q

How is Elisa different in the way you can quantify protein

A

Specifically quantifies using secondary ab which is conjugated with an enzyme causing colour change not chemilinescent

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13
Q

What is sandwich Elisa for protein quantification in cell lysate

A

Capture ab attached to well and then lysate added first then primary and secondary

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14
Q

What is required to be made to determine conc from spectrophotometer od readings

A

A standard curve of known conc

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15
Q

How many replicates would you do on the micro tire plate

A

3 for each conc and 3 for each sample eg treated and non treated

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16
Q

What do you draw from the standard curve which you use to determine conc

A

A line of best fit- NOT JOINING DOTS

17
Q

Right now you don’t know if synaptophysin is causing differentiation or if diff is causing synaptophysin exp. How do you test if synaptophysin affects differentiation in neural sc

A

Use siRna which complexes with risc, causing cleavage and degradation of the transcript therefore reduced protein levels

Using immunofluorescence of cells treated with gf you can see whether this has caused a lack of differentiation

18
Q

What would need to be the control to make sure siRna is reducing synaptophysin nothing else (on western blot) - distinguish non-specific degradation from specific

A

Scrambled siRna which is non targeting siRna would be used as a control

Shouldn’t affect synaptophysin levels

19
Q

Neural sc treated with scrambled siRna would show whag under immunofluorescence

A

Full differentiation if treated with synaptophysin