Non Genetic Analysis of Gene Function

Question 1

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

Answer 1

Where is the protein

Where is it transcribed

Question 2

How is a protein specific antibody made?

Answer 2

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

Question 3

Why do expression plasmids often include an epitope tagging system?

Answer 3

It allows rapid and efficient purification of the protein

The tag is fused in frame to the cDNA during cloning

Question 4

What are epitope tags?

Answer 4

Peptides for which antibodies are already available

Question 5

Describe how antibody-affinity purification works

Answer 5

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

Question 6

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

Answer 6

An epitope

Question 7

What is a tagged antibody?

Answer 7

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

Question 8

How are specific antibodies made?

Answer 8

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

Question 9

List commonly used enzyme conjugates

Answer 9

Alkaline phosphatase - substrate turns blue

Horseradish peroxidase - substrate turns brown

Question 10

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

Answer 10

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

Question 11

How many antibodies do we need to amplify a signal?

Answer 11

2 as many secondary antibodies bind to each primary antibody

Question 12

How are antibodies used to visualise proteins?

Answer 12

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

Question 13

Why do we use formaldehyde?

Answer 13

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

Question 14

How is RNA in situ analysis performed?

Answer 14

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

Question 15

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

Answer 15

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

Question 16

Give the wavelength of light which excites GFP

Answer 16

475nM

Question 17

Give the wavelength of light the GFP emmits

Answer 17

510nM

Question 18

Which species does GFP come from?

Answer 18

Aequorea victoria (jellyfish)

Question 19

Give the steps used to create a transgenic line

Answer 19

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

Question 20

List uses for GFP transgenic lines

Answer 20

• 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