Considerations before starting your IP experiment

Learn the importance of antibody titration in order to determine the optimal signal-to-noise ratio in IP.

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Antibody Titration for Immunoprecipitation

A titration experiment should be performed with multiple antibody concentrations in order to determine the optimal signal-to-noise ratio. In general, this titration should range from 1 to 10.0 μg for ~5000 μg of protein extract.


As an example, if a datasheet suggests a 1:100 dilution, the titration experiment should include dilutions of 1:25, 1:50, 1:100, 1:200, and 1:250. To keep the same experimental conditions, after selecting the incubation time, all dilutions should be performed with the same type of sample.


Isotype & Negative Control Bands

Isotype Control

An isotype control is used to determine which bands in the experimental sample are specific versus non-specific signal due to the isotype. The isotype control should always be run in parallel to the sample.

Negative Control

Plain beads (without antibody) can be used as negative controls. They help to distinguish specific and non-specific bindings. Any products obtained with these control conditions can be attributed to non-specific (off-target) interactions.


Protein Isoforms, Post-Translation Modification & Protein Interaction


The STRING database ( aims to provide a critical assessment and integration of protein–protein interactions, including direct (physical) and indirect (functional) associations. It covers more than 2000 organisms, which necessitates novel, scalable algorithms for transferring interaction information between organisms; based on genomic data, and high throughput analyses including co-expression experiments.

(UnitProt database; (, PubMed https://


Phosphatase, Methylation & Deubiquitination Inhibitors; Why They Are Important

Preservation of protein post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, or methylation can be required to maintain protein–protein interaction. They play essential roles in regulating chromatin dynamics. Maintaining PTMs during cell lysis with phosphatase, methyltransferase, or deubiquitinase inhibitors can be important for the maintenance of in vivo interactions in vitro. As an example, adding phosphatase inhibitors to the lysis buffer will maintain the phosphorylated stage of the protein of interest.

Table 2 shows a few examples of phosphatase, methylation, and deubiquitination inhibitors.

Table 2
Phosphatase Inhibitors Action
Sodium Orthovandate with the correct pH Tyrosine phosphatase inhibitor
β-Glycerophosphate Serine and threonine phosphatase inhibitor
Okadaic Acid Protein phosphatase 1/2A inhibitor
Sodium Fluoride Serine and threonine phosphatase inhibitor
Deubiquitination Inhibitors Action
TAME Ubiquitin ligase inhibitor
USP14 Inhibitor, IU1 USP14 deubiquitinase (DUB) inhibitor
DUB Inhibitor IV, b-AP15 19S DUB inhibitor
DUB Inhibitor VI, P22077 USP7 and USP47 DUB inhibitor
Methylation Inhibitors Action
RG108 DNA methyltransferase (DNMT) inhibitor
5-Azacytidine DNMT inhibitor