Overview of Immunoprecipitation (IP)

Jump to Antibodies

 

IP, or immunoprecipitation, is a highly adaptable laboratory technique used to target and isolate a specific protein from a mixed solution.

 

What is IP?

 

Immunoprecipitation uses antibodies to bind and isolate a specific protein from a mixed biologic or biochemical solution. Many variants on immunoprecipitation also exist, including co-IP--to identify the protein binding partners of the target protein1, and ChIP--to determine where in the genome a DNA-binding protein binds2. In IP, a protein of interest is targeted with a specific antibody to precipitate it out of solution. A small quantity of protein is ultimately recovered and can be used for semi-quantitative analysis. ChIP, co-IP, and other variants of the technique may also pull down bound proteins or genetic material, which can be further analyzed after the precipitation step.

 

How Does It Work?

 

The principle of immunoprecipitation relies on the recognition of a target protein antigen by a specific antibody. In order for the antibody-protein complex to precipitate out of solution, it must be bound to a solid state material. This can be done in one of two ways: either the antibody is bound to agarose or magnetic beads linked to protein A or G and then added to the protein-containing solution, or free antibody is added to the solution containing the target protein and the antibody-protein complex is subsequently bound to beads3.

Regardless of the method chosen to target the protein of interest, the technique used for the precipitation is a function of the type of beads used. If magnetic beads are used, the tube containing the sample mixture can be placed in a magnetic chamber to capture the immunoprecipitate, and the unbound proteins can be washed away while the target protein remains. If agarose beads are used, the sample can be centrifuged to pellet the beads bound to the target protein, washed, and the supernatant discarded.

 

Why IP?

 

IP can be used as the first step in larger experiments that require isolating individual proteins or protein-containing complexes4. When the goal is to isolate individual proteins, IP can be used for small-scale protein purifications, such as isolating a target protein produced by genetically engineered cells. It can also be used to enrich the concentration of a protein target that may be found in low abundance in the primary solution. Proteins immunoprecipitated out of solution can be further analyzed for their molecular weight or post-translational modifications by using techniques such as western blotting or mass spectrometry. Protein-protein interactions can be characterized using co-IP or RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) which use IP followed by western blotting or mass spectrometry to identify characteristics of two or more complexed proteins5. Besides its utility in isolating proteins and protein complexes, techniques including ChIP, ChIP-seq, and RIP (RNA immunoprecipitation) can be used to map the nucleotide binding capacity of particular proteins in biologic samples6. Additionally, IP can play a crucial role in yeast two-hybrid assays, since it can be used as a secondary method to control for the specificity of the technique7.

 

Recent Immunoprecipitation Citations Using Bethyl Antibodies

 

Bethyl Laboratories sells high quality antibodies for use in immunoprecipitation. For example, these products have been used recently to:

  • Isolate proteins related to protein translation in E. coli8
  • Study the regulation of epigenetic methylation in the eye9
  • Identify binding between downstream members of the Akt signaling pathway10
  • Characterize the regulation of inflammation and vascular pathologies11

 

 

Thousands of Bethyl antibodies are validated for use in immunoprecipitation. The complete list is here:

 

IP

A20
AC9
ACD
ACL
ACS
ADA
ADK
AE2
AF4
AF6
AF9
AIF
AIP
Aly
AN
AP4
APC
AQR
AR
ARG
ASF
ATM
ATR
AU1
AU5
Axl
AZ1
C3G
CAD
CAL
CAR
CAT
CBL
CBP
CBS
CD4
CD5
CD7
CEE
CGN
CHC
CIA
CIC
CIT
CIZ
CPD
CR2
CSB
CSK
MAX
MAZ
MFF
MGA
MK5
MNT
MOF
MPI
MVP
MYB
MYC
p17
P23
p49
p53
P55
p66
p73
p75
PA1
PAF
PB1
PBE
PBK
Pc2
PC4
PF1
PGD
PGP
PGR
PKM
PML
PNN
PNP
PPL
PSF
PXK
PXN
PZR
RAN
Rb1
RCL
RCP
REA
RED
REL
RIG
RLF
RMP
ROD
RP1
SA1
SA2
SCD
SET
SF1
SFN
SIL
SIP
SKI
SKT
SLK
Sp1
SPN
SPP
SRA
SRF
SRM
SSB
SYK
T7
TDG
TFG
TG2
TK1
TPR
TR2
TS
TSN
TTK
TZF
References

1. Collas, P. 2010. The current state of chromatin immunoprecipitation. Mol Biotechnol. May;45(1): 87-100.

2. Mahony S, Pugh, BF. 2015. Protein-DNA binding in high resolution. Crit Rev Biochem Mol Biol. 50(4): 269-283.

3. Kaboord B. and Perr M. 2008. Isolation of proteins and protein complexes by immunoprecipitation. Methods Mol Biol.424:349-364.

4. Stefanie Boellner, Karl-Friedrich Becker. 2015. Recent progress in protein profiling of clinical tissues for next-generation molecular diagnostics. Expert Review of Molecular Diagnostics. 15(10):1277-1292.

5. Rao, VS, Srinivas K., Sujini GN, Kumar, GN. 2014. Protein-protein interaction detection: methods and analysis. Int J Proteomics. 2014:147648.

6. Wade JT. 2015. Mapping transcription regulatory networks with ChIP-seq and RNA-seq. Adv Exp Med Biol. 883:119-134.

7. Steinbrenner J, Eldridge M., Tomé DFA., Beynon JL. 2014. A Simple and Fast Protocol for the Protein Complex Immunoprecipitation (Co-IP) of Effector: Host Protein Complexes. Methods Mol Biol. 2014;1127:195-211.

8. Byrgazov K, Grishkovskaya I, Arenz S, Coudevylle N, Temmel H, Wilson DN, Djinovic-Carugo K, Moll I. 2015. Structural basis for the interaction of protein S1 with the Escherichia coli ribosome. Nucleic Acids Res. Jan;43(1):661–673.

9. Sun J, Zhao Y, McGreal R, Cohen-Tayar Y, Rockowitz S, Wilczek C, Ashery-Padan R, Shechter D, Zheng D, Cveki A. 2016. Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers. Epigenetics & Chromatin. Sept 9;9(1):37.

10. Zeng M, van der Donk WA, Chen J. 2014. Lanthionine synthetase C-like protein 2 (LanCL2) is a novel regulator of Akt. Mol Biol Cell. Dec 1;25(24):3954-3961.

11. Yang Y, Cheng X, Tian W, Zhou B, Wu X, Xu H, Fang F, Fang M, Xu Y. 2014. MRTF-A steers an epigenetic complex to activate endothelin-induced pro-inflammatory transcription in vascular smooth muscle cells. Nucleic Acids Res. 42(16):10460–10472.