Affinity chromatography Flashcards
Overview
how AC works
group-specific ligand
examples of group-specific ligands
Immobilized vs indirectly coupled ligands
elution/ wash buffers
Common elution buffer system
the issue of crapome
AC of tagged P
fusion tag purification
add tag to C terminal for better chance of extracting a full length protein and reduce protease activity on target
His tag
IMAC
IMAC suite
IMAC for insoluble proteins
Denatured protein can be used as an antigen for antigen-antibody analysis
can be dissolved on SDS and ran on gel, then transferred to membrane for Western/ overlay.
Far western overlay can be used to identify protein-protein interactions and interacting motif from SDS PAGE
Different types of AC
Peptide AC
Immunoprecipitation AC
Co-immunoprecipitation
Co-immunoprecipitation of Kif18a and PP1
Pull-down assays
Pull-down assay example: Kif18a and PP1
Displacement AC
Displacement AC example
displacement AC example suite
Kif18 is a kinesin (motor protein) that takes PP1 to the mitotic spindle kinetochore
TAP AC
Overview of TAP method
C terminus usually have specialized function.
Unsure how TAP will affect protein fold. TAP may prevent some interacting proteins from binding for that reason the position of the tag is altered.
TAP strategy
Results
Kinobeads
Relevance
background
kinobeads pull-down
relevance of study
results
conclusion
