Protein interactions

Question 1

4 examples of motifs DNA binding proteins might incorporate

Answer 1

1. Leucine zipper motifs
2. Zinc fingers
3. basic helix-loop-helix
4. Beta sheet

Question 2

How can DNA binding proteins be extracted from other proteins?

Answer 2

Affinity chromatography- beads coated in oligonucleotides

Question 3

What is a gel mobility shift assay used for?

Answer 3

Used to study protein-DNA interactions

Bands on the gel tell you if a protein interacts with DNA or not

Question 4

What does a DNA footprint show?

Answer 4

Where a DNA binding protein has prevented the DNA from being cleaved by DNase

Question 5

What is a EF hand domain?

Answer 5

Two alpha helices linked by a short loop region that binds calcium

Question 6

What does a SH2 domain do?

Answer 6

Binds to phosphorylated tyrosine residues

Question 7

What does a SH3 domain do?

Answer 7

Binds to proline rich motifs/ adaptor proteins

Question 8

What does the PH domain do?

Answer 8

Binds to phosphorylated lipids- anchors proteins to the membrane

Question 9

What are the main proteins involved in insulin signalling?

Answer 9

Insulin receptor tyrosine kinase
Insulin receptor substrate (IRS1)
Grb2- Adaptor protein
SOS
Scaffold protein

Question 10

How can we biochemically study protein-protein interactions?

Answer 10

Chromatography
Pull down reactions
Immunoprecipitation

Question 11

What happens in immunoprecipitation?

Answer 11

Specific antibodies will bind to the tagged protein, beads coated in protein A will bind to these antibodies, thus precipitating the protein complexes

Question 12

Common tags used in immunoprecipitation

Answer 12

HA peptide
Myc peptide
Flag peptide

Question 13

What happens in the GST affinity pulldown?

Answer 13

GST tag on proteins will bind to glutathione coated beads

Glutathione solutions elute the proteins from the beads

Question 14

Commons tags in pulldown reactions

Answer 14

GST

6xHis

Question 15

What is a yeast two-hybrid screen?

Answer 15

Method to see whether 2 proteins interact with one another
Target protein- bound to DNA binding domain
Binding protein- bound to transcriptional activation domain
Transcription of the essential gene only occurs if the 2 proteins interact with each other

Question 16

What are 5 ways of measuring the strength of protein interactions?

Answer 16

Elisa
Radioactive binding assay
Fluorescence resonance energy transfer
Surface plasmon resonance 
Live imaging