OC4 - determination of chemical bonding at the binding site

Question 1

antibody trapping

Answer 1

involves raising antibodies against a purified protein in order to analyse it.
specific antibodies are in the column, and when the protein sample is passed through, the antibodies bind to the target protein whilst the non-target protein passes through. A buffer is added and the target proteins are then eluted and can be analysed by 2D gel electrophoresis and mass spectrometry

Question 2

FRET (fluorescence resonance energy transfer)

Answer 2

allows analysis of proteins to determine if they associate with one another through the use of fluorescence
dependent on one fluorescence protein absorbing fluorescent energy from another
two proteins of interest are tagged with complementary fluorescent molecules, if the tagged proteins interact there will be a change in fluorescent wavelength.

Question 3

yeast 2 hybrid assay

Answer 3

uses two domains in the transcriptional activator protein :
DNA binding domain, and the activation domain - both are needed for translation
one protein of interest - bait- is fused to the DNA binding domain
the other protein of interest -fish- is fused to a transcription activation domain
if the two proteins interact a functional transcriptional activator is produced which activates a reporter gene which produces a coloured product showing an interaction has taken place

Question 4

affinity tag separation

Answer 4

the target protein is tagged with an affinity tag to produce a recombinant protein, which is passed through a chromatography column that is lined with a substrate; the target protein will bind to the substrate, and, other non-specific proteins will be eluted.
the sample is washed with a wash buffer to remove any unspecific proteins, then an elution buffer is used. the elution buffer is designed to disrupt the binding, allowing the recombinant (target) protein to be released from the column and eluted for analysis.