L16 - Non-genetic Analysis of Gene Function Flashcards
What do you use to tell where a protein is in an organism?
Antibodies
What do you use to tell where a gene is transcribed in an organism?
RNA in situ
What do you use to visualise gene expression and protein localisation in living cells?
GFP
How to make a protein specific antibody overview
Use protein to immunize animal (rabbit)
Generates immune response - antibodies
Making a protein specific antibody full method
- Need to make a lot of the protein
- Expression vectors use a bacteriophage promoter to drive high levels of RNA synthesis
a. Promoters have to be inducible (can turn them off and on) otherwise bacteria die rapidly - Chemicals or temperature shifts are used to induce protein expression
- Bacteria harvested in a centrifuge and lysed to make a crude extract
- Expression plasmids often include an epitope tagging system
a. Allows for rapid and efficient purification of protein - Tag is fused in-frame to the cDNA during the cloning – helps purify the protein
What are epitope tags?
Peptides for which antibodies are already available
Antibodies bind to small regions of proteins with very high specificity - epitope
Antibody-affinity purification method
- Bacterial cells are lysed and the resulting solution (crude extract) is poured onto a column
- Column contains small beads coated with antibody - recognise the epitope tag which contains protein of interest
- Tagged antibodies have dyes or enzymes attached so we can determine their location
- Protein eluted for column using a pH 3 buffer
What are the 4 detection methods?
Fluorescent dyes
Antibodies
Enzyme conjugates
Two antibody sandwhich
Detection - fluorescent dyes
Allow us to detect location using specific wavelenghs of light
Detection - antibodies
Examine the sub-cellular localization of the protein
Testing where the protein is in the organism
Detection - enzyme conjugates
Alkaline phosphatase - substrate turns blue
Horseradish peroxidase - substrate turns brown
Enzyme detection can enhance sensitivity
Detection - two antibody sandwhich
Amplifies the signal
Many 2º antibodies bind to each 1º antibody
1º antibody made in rabbit
2º antibody made in mouse - binds rabbit antibodies and carries the tag
How to use antibodies method
- Chemically fix (formaldehyde) animal or tissue to stabilize the structure (whole structure or slice)
- Incubate with tagged antibody
- Wash of excess antibody
- Whole or slice mount
RNA in situ analysis method
- Purified vector containing the cDNA of interest (template)
- Synthesise RNA antisense probe - incorporate epitope tagged nucleotides
- Incubate embryo with antisense probe
- Antisense probe hybridises with the endogenous mRNA
- Wash off excess probe
- RNA detection systems use a blue substrate
Bicoid in situ vs antibody
Whole mount analysis of Drosophila embryo
mRNA highly concentrated at anterior end
GFP excitation
475 nM
GFP emission
510 nM
Generating a GFP transgenic line – transgenesis method
- Clone the entire gene into a plasmid
- Genetically engineer GFP onto the end of the last exon or replace the gene (reporter construct)
- Integrate the GFP fusion gene back into organism’s genome
o Involves microinjecting a solution of the DNA into the one-cell zygote
o DNA then randomly integrates into genome - Integrated gene - transgene
What are the 3 uses of GFP transgenic lines?
To follow expression of a gene in the animal
To follow subcellular localisation of a protein
To follow the behaviour of cells in vivo
GFP transgenic lines - follow expression of gene in animal
To see which tissues gene is expressed in
GFP transgenic lines - follow subcellular localisation of a protein
Some transcription factors become nuclear upon activation
This happens after the cell receives a signal - use a GFP fusion
GFP fusion does not usually alter the activity of the protein
GFP transgenic lines - follow the behaviour of cells in vivo
GFP line used to mark specific cells so they are distinguishable - use a GFP reporter construct
