Immunoprecipitation (IP)

Question 1

What is Immunoprecipitation (IP)?

Answer 1

Technique used to isolate and purify specific proteins using antibodies

Question 2

What is the starting material for Immunoprecipitation (IP)?

Answer 2

Cell lysate

Question 3

What is added instead of secondary antibodies during Immunoprecipitation (IP)?

Answer 3

Adapose beads

Question 4

What do agarose beads bind to in IP?

Answer 4

Bind to Fc (constant regions) of primary antibody

Question 5

What do agarose beads contain in IP?

Answer 5

Agarose
Bacterial protein A or G (optional)
Magnetic centre (optional)

Question 6

What are the two methods of separation during IP?

Answer 6

1. Centrifuging: Pellet at bottom will contain beads-antibody-protein complex. Use normal beads.
2. Magnetic rack: Magnetic field pulls the beads toward the side of the tube.

Question 7

What is Co-Immunoprecipitation (Co-IP) used to identify?

Answer 7

Protein-protein interactions

Question 8

In which step does Co-IP differ from IP?

Answer 8

Lysis step
Extra care is given as not to disrupt protein-protein interactions.

Question 9

After elution, how many bands are there on SDS-PAGE in IF compared to Co-IF?

Answer 9

IF: 1
Co-IP: multiple (protein of interest and proteins it is bound to)

Question 10

What is
Chromatin ImmunoPrecipitation sequencing (ChIP-seq)?

Answer 10

Technique used to investigate protein-DNA (to find exact binding site)

Question 11

What is ChIP-seq a combination of?

Answer 11

Chromatin immunoprecipitation and next-generation sequencing (NGS)

Question 12

In ChIP-seq, what are the DNA binding proteins and DNA cross-linked with?

Answer 12

formaldehyde (to form covalent bond)

Question 13

What happens in ChIP-seq after cross-linking?

Answer 13

DNA is fragmented (proteins still bound)