Affinity chromatography

Question 1

Overview

Answer 1

Question 2

how AC works

Answer 2

Question 3

group-specific ligand

Answer 3

Question 4

examples of group-specific ligands

Answer 4

Question 5

Immobilized vs indirectly coupled ligands

Answer 5

Question 6

elution/ wash buffers

Answer 6

Question 7

Common elution buffer system

Answer 7

Question 8

the issue of crapome

Answer 8

Question 9

AC of tagged P

Answer 9

Question 10

fusion tag purification

Answer 10

add tag to C terminal for better chance of extracting a full length protein and reduce protease activity on target

Question 11

His tag

Answer 11

Question 12

IMAC

Answer 12

Question 13

IMAC suite

Answer 13

Question 14

IMAC for insoluble proteins

Answer 14

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

Question 15

Different types of AC

Answer 15

Question 16

Peptide AC

Answer 16

Question 17

Immunoprecipitation AC

Answer 17

Question 18

Co-immunoprecipitation

Answer 18

Question 19

Co-immunoprecipitation of Kif18a and PP1

Answer 19

Question 20

Pull-down assays

Answer 20

Question 21

Pull-down assay example: Kif18a and PP1

Answer 21

Question 22

Displacement AC

Answer 22

Question 23

Displacement AC example

Answer 23

Question 24

displacement AC example suite

Answer 24

Kif18 is a kinesin (motor protein) that takes PP1 to the mitotic spindle kinetochore

Question 25

TAP AC

Answer 25

Question 26

Overview of TAP method

Answer 26

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.

Question 27

TAP strategy

Answer 27

Question 28

Results

Answer 28

Question 29

Kinobeads

Answer 29

Question 30

Relevance

Answer 30

Question 31

background

Answer 31

Answer 32

Question 33

kinobeads pull-down

Answer 33

Question 34

relevance of study

Answer 34

Question 35

results

Answer 35

Question 36

conclusion

Answer 36