全部

全部
产品管理
新闻资讯
介绍内容
企业网点
常见问题
企业视频
企业图册

Everyone is searching:
Nanoantibody screening

Yeast Two-Hybrid Y2H
Mb Y2H for Membrane Proteins

Yeast one-Hybrid Y1H
TF-centered YlH
Yeast Three-Hybrid Y3H

Abiotic-Stress Resistance Gene Screening

Digital Analog Library Screening

ABOUT US

Empowering Science with Precision and Simplicity!

Protein-Protein Interaction Research Tool: A Comprehensive Analysis of Co-IP

Release time:

2025-02-05

Co-IP (Co-immunoprecipitation)

Principle

The principle of Co-IP is that if two proteins specifically interact in vitro, then when an antibody against one of the proteins is used for immunoprecipitation, the other interacting protein will also be precipitated. The fundamental concept is based on the specific binding between antigen and antibody, and the goal is to detect protein-protein interactions.

Figure 1: Co-IP Principle

Experimental Steps
The Co-IP experimental process can be generally summarized in the following four steps:

1.Protein Extraction – Obtain a mixture of proteins by lysing cells.

2.Incubation – Mix the protein sample with an antibody specific to the target protein.

3.Elution – Elute the proteins that have been pulled down.

4.Detection – Detect the interacting protein by using an antibody against the partner protein in the precipitate.

Application Case Studies
In the paper "Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector," the authors aimed to elucidate the molecular mechanism behind the immune suppression mediated by ROD1. To validate the proteins interacting with ROD1, they initially used a yeast two-hybrid (Y2H) screening. To confirm the interactions, the authors transiently expressed ROD1, APIP6, and ARIP1 in Nicotiana benthamiana. ROD1 was tagged with GFP, and the two E3 ubiquitin ligases (APIP6 and ARIP1) were tagged with Myc. Negative controls were also included by expressing ROD1 alone or APIP6/APIP1 alone, which showed no interaction, confirming proper experimental setup.

In the input group, bands confirmed the presence of ROD1, APIP6, and APIP1 in the protein samples. After co-expressing ROD1 and APIP6, the proteins were pulled down using Myc antibody beads (IP group, lane 3). Western blotting (WB) with ROD1 and APIP6 antibodies showed bands, confirming that APIP6 could pull down ROD1 and that the two proteins interact. Using the same method, ROD1 was also shown to interact with APIP1.

Figure 2: Co-IP Detection of ROD1 Interaction with RIP1 and APIP6 in Nicotiana benthamiana Leaves

In addition, Y2H screening identified a catalase enzyme, CatB, as a potential interacting partner of ROD1. To confirm this interaction, Co-IP was again used. The known NLR protein PigmR (PigmR-CC) was used as a negative control because its coiled-coil (CC) domain does not interact with CatB. In the input group, several proteins were confirmed to be present in the protein samples. In the IP group, PigmR-CC did not interact with ROD1, and no complex was pulled down by GFP antibody beads, confirming the negative result. In contrast, in the final IP group, a protein band was detected for both ROD1 and CatB, confirming their interaction.

Figure 3: Co-IP Detection of ROD1 and CatB Interaction

From these case studies, we can see that Co-IP experimental result analysis is not overly difficult. The key is to understand the experimental principles, procedures, and controls, and to carefully analyze the results through practical examples.

Related News

2025-07-16

Literature Sharing | Interaction of PsMYB4 with PsEGL3 inhibits anthocyanin biosynthesis in tree peony yellow flowers

This study explores the molecular mechanism behind yellow flower formation in tree peony, a highly valued ornamental plant in China. Researchers identified two transcription factors, PsMYB4 and PsEGL3, that are highly expressed in a yellow-flowered cultivar.

2025-07-10

Literature Sharing | Putative Upstream Regulators DoNF-YB3 and DoIDD12 Correlate with DoGSTF11 Expression and Anthocyanin Accumulation in Dendrobium officinale

This study explores the role of the DoGSTF11 gene in Dendrobium officinale, a traditional medicinal herb. While previous research has focused on polysaccharides and alkaloids, this work addresses the lesser-known anthocyanin pathway. The researchers found that DoGSTF11 is highly expressed in the purplish variety of D. officinale and is localized in the nucleus and cell membrane, though it lacks transcriptional activation ability. Overexpressing DoGSTF11 in tomato led to increased anthocyanin accumulation, suggesting it plays a role in anthocyanin transport or sequestration. Protein interaction studies revealed that DoGSTF11 interacts with DoGST31, and that DoIDD12 and DoNF-YB3 may regulate its expression. Overall, the findings highlight DoGSTF11 as a positive regulator of anthocyanin accumulation and offer new insights for flavonoid metabolic engineering in D. officinale.

2025-07-08

Literature Sharing | Chinese cabbage orphan gene BR3 confers bolting resistance to Arabidopsis through the gibberellin pathway

Premature bolting affects yield and quality in Chinese cabbage, highlighting the importance of identifying bolting resistance genes. This study identifies an orphan gene, BR3 (BOLTING RESISTANCE 3), in Arabidopsis thaliana as a positive regulator of bolting resistance. BR3 is expressed during the seedling and flowering stages and localizes to the plasma membrane and nucleus. Overexpression of BR3 (BR3OE) delays bolting and flowering under both long-day and short-day conditions, with increased rosette leaf number and reduced plant height. Key flowering genes are downregulated in BR3OE plants. GA₃ treatment induces early flowering in both BR3OE and wild type (WT) plants, but BR3OE still flowers later than WT. In Chinese cabbage, BR3 shows co-expression with DELLA genes BrRGA1 and BrRGL3, suggesting a regulatory role through the GA pathway. This study offers new insights into bolting resistance mechanisms and provides valuable targets for breeding bolting-resistant Chinese cabbage varieties.

2025-07-03

Literature Sharing | Regulation of co-translational mRNA decay by PAP and DXO1 in Arabidopsis

This study investigates the regulation of the co-translational mRNA decay (CTRD) pathway in Arabidopsis, a critical mechanism for maintaining mRNA homeostasis. The researchers found that 3ʹ-phosphoadenosine 5ʹ-phosphate (PAP), an inhibitor of exoribonucleases, affects CTRD activity. Specifically, they showed that loss of FRY1 impairs XRN4-dependent CTRD and that exogenous PAP treatment stabilizes CTRD target mRNAs. Additionally, they discovered that another PAP-sensitive exoribonuclease, DXO1, also contributes to CTRD, likely by acting on NAD⁺-capped mRNAs involved in stress responses. These findings reveal new layers of regulation and additional players in the CTRD pathway in plants.

2025-07-01

Literature Sharing | Two pathogen-inducible UDP-glycosyltransferases, UGT73C3 and UGT73C4, catalyze the glycosylation of pinoresinol to promote plant immunity in Arabidopsis

This study uncovers a novel immune regulatory pathway in Arabidopsis thaliana involving two UDP-glycosyltransferases, UGT73C3 and UGT73C4, which are highly induced by Pseudomonas syringae infection. Overexpression of these genes enhances disease resistance, while their double mutation compromises immunity. Metabolomic analysis and biochemical assays reveal that UGT73C3/C4 glycosylate pinoresinol into its mono- and diglucoside forms, which promote immune responses by boosting ROS production and callose deposition. Additionally, the transcription factor HB34 directly activates UGT73C3/C4 expression, linking transcriptional regulation to glycosylation-mediated immunity. This work highlights the physiological significance of UGTs in plant defense through pinoresinol glycosylation.

2025-06-27

Literature Sharing | PbARP1 enhances salt tolerance of ‘Duli’ pear (Pyrus betulifolia Bunge) through abscisic acid signalling pathway

This study investigates salt stress tolerance in pears and identifies 35 salt-tolerant genes using a yeast expression library. Among them, PbARP1 was found to be significantly upregulated under salt stress in 'Duli' pear (Pyrus betulaefolia). Functional analyses showed that overexpression of PbARP1 in transgenic pear calli enhanced salt tolerance, while its suppression increased sensitivity to salt stress. Silencing PbARP1 also altered the expression of key ABA signaling genes, including PbPYL4, PbPYL9, PbPYL8, PbSRK2I, and PbABI5, suggesting that PbARP1 modulates salt stress responses through the ABA signaling pathway. Furthermore, PbPYL8, an ABA receptor, was identified as a protein interacting with PbARP1, highlighting its pivotal role in ABA-mediated salt stress regulation in pear.

2025-06-24

Literature Sharing | Phosphorylation of the strawberry MADS-box CMB1 regulates ripening via the catabolism of abscisic acid

This study uncovers a regulatory mechanism linking transcriptional control and posttranslational modification in strawberry fruit ripening. The MADS-box transcription factor FaCMB1 acts as a negative regulator of ripening, with both its transcript and protein levels decreasing during fruit development, a process enhanced by ABA. Functional manipulation of FaCMB1 significantly affected ripening and ABA content.

2025-06-20

Literature Sharing | Overexpression of soybean flavonoid 3′-hydroxylase enhances plant salt tolerance by promoting ascorbic acid biosynthesis

This study reveals that the flavonoid 3′-hydroxylase gene GmF3′H plays a key role in enhancing salt tolerance in soybean by regulating redox homeostasis. Using CRISPR/Cas9 knockout and overexpression approaches, the researchers demonstrated that GmF3′H competitively interacts with CSN5B, disrupting its binding to VTC1, a key enzyme in ascorbic acid biosynthesis. This redirection of metabolic flux toward the L-galactose pathway leads to increased ascorbic acid (AsA) levels, enhancing ROS scavenging capacity and improving salt stress tolerance during seed germination and seedling growth.

Do you have a question for us?

contact our experts

Explore More →

Tel: +86 025-85205672

Email: info@pronetbio.com

Address: Building 3C, Nanjing Xianlin Zhigu,

Qixia District, Nanjing, Jiangsu, China, 210033

Subscribe newsletter

Business license

Need more info?

Let's connect!

Any question? Get in touch with us!