Non Genetic Analysis of Gene Function Flashcards Preview

Cell and Molecular Biology > Non Genetic Analysis of Gene Function > Flashcards

Flashcards in Non Genetic Analysis of Gene Function Deck (20)
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1
Q

What can you find out with non genetic analysis of a gene?

A

Where is the protein

Where is it transcribed

2
Q

How is a protein specific antibody made?

A

1) Expression vectors use a bacteriophage promoter to drive RNA synthesis
2) Chemical or temperature shifts induce protein expression
3) Bacteria are harvested in a centrifuge and lysed to collect the extract

3
Q

Why do expression plasmids often include an epitope tagging system?

A

It allows rapid and efficient purification of the protein

The tag is fused in frame to the cDNA during cloning

4
Q

What are epitope tags?

A

Peptides for which antibodies are already available

5
Q

Describe how antibody-affinity purification works

A

Bacterial cells are lysed and the crude extract is poured in a column
The column contains antibodies attached to small beads
Proteins recognised by the antibody remain in the column whilst the others are lost
The bound protein is then removed from the antibody beads by eluting with a pH3 buffer

6
Q

What is the part of a protein which binds to an antibody called?

A

An epitope

7
Q

What is a tagged antibody?

A

An antibody with a dye or enzyme attached to them so we can determine their location

8
Q

How are specific antibodies made?

A

Grow bacterial culture and purify the protein
Inject rabbit several times over a 3 month period
Purify specific antibody from the serum

9
Q

List commonly used enzyme conjugates

A

Alkaline phosphatase - substrate turns blue

Horseradish peroxidase - substrate turns brown

10
Q

Where is the secondary antibody made if the primary antibody is made in a rabbit?

A

In a mouse - it binds to rabbit antibodies and carries the tag

11
Q

How many antibodies do we need to amplify a signal?

A

2 as many secondary antibodies bind to each primary antibody

12
Q

How are antibodies used to visualise proteins?

A

1) Chemically fix with formaldehyde the tissue or animal
2) Incubate with tagged antibody
3) Wash off excess antibody

13
Q

Why do we use formaldehyde?

A

It stabilises the structures within the cell by forming cross links between them

14
Q

How is RNA in situ analysis performed?

A

1) Purify vector containing cDNA of interest
2) Synthesise RNA antisense probe; incorporating epitope tagged nucleotides
3) Incubate embryo with antisense probe
4) Antisense probe hybrdises with the endogenous mRNA
5) Wash off the excess probe
6) RNA detection system use a blue substrate

15
Q

Describe the difference of bicoid in situ vs antibody staining in the fruit fly drosophila before cellularisation

A

In situ - bicoid mRNA is localised to the maternal end of the embryo
Bicoid protein is seen in a gradient

16
Q

Give the wavelength of light which excites GFP

A

475nM

17
Q

Give the wavelength of light the GFP emmits

A

510nM

18
Q

Which species does GFP come from?

A

Aequorea victoria (jellyfish)

19
Q

Give the steps used to create a transgenic line

A

1) Clone the entire gene (genomic DNA) with all of the regulatory elements in to the plasmid
2) Genetically engineer GFO onto the end of the last exon (gene fusion) or replace the gene (reporter construct)
3) Integrate the GFP fusion gene back in to the organiss genome by microinjecting the the DNA into the one cell zygote so the DNA randomly integrates with the genome

20
Q

List uses for GFP transgenic lines

A
  • Looking at microtubules in cell division
  • To follow expression of a gene
  • To follow subcellular localisation of a protein
    To follow the behaviour of cells in vivo