If a control test with only probes and no antibody shows high background, the cause is likely non-specific binding of the probes. To address this issue, adding sonicated salmon sperm DNA (Product No. D9156 - Deoxyribonucleic acid, single stranded from salmon testes) at 2.5 ug/ml to the blocking buffer may help reduce this background.
DUO92202
Duolink® PLA Control Kit - PPI
Control Kit to reliably detect Protein-Protein Interactions (PPI) with Duolink® PLA
Synonym(s):
in situ Proximity Ligation Assay Control Kit, Protein Protein Interaction Assay Control Kit
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About This Item
UNSPSC Code:
41105331
NACRES:
NA.84
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product line
Duolink®
technique(s)
immunofluorescence: suitable
proximity ligation assay: suitable
suitability
suitable for fluorescence
shipped in
dry ice
storage temp.
−20°C
1 of 4
This Item | DUO96020 | DUO96000 | DUO96030 |
|---|---|---|---|
| technique(s) immunofluorescence: suitable, proximity ligation assay: suitable | technique(s) immunofluorescence: suitable, multiplexing: suitable, proximity ligation assay: suitable | technique(s) immunofluorescence: suitable, multiplexing: suitable, proximity ligation assay: suitable | technique(s) immunofluorescence: suitable, multiplexing: suitable, proximity ligation assay: suitable |
| suitability suitable for fluorescence | suitability suitable for fluorescence | suitability suitable for fluorescence | suitability suitable for fluorescence |
| product line Duolink® | product line Duolink® | product line Duolink® | product line Duolink® |
| shipped in dry ice | shipped in dry ice | shipped in dry ice | shipped in dry ice |
| storage temp. −20°C | storage temp. −20°C | storage temp. −20°C | storage temp. −20°C |
General description
Duolink® PLA Control Kits are intended for use with Duolink® PLA reagents to ensure proper function of reagents and Duolink® PLA staining technique.
Application
Application note
In the Duolink® PLA Control Kit, the cells and antibodies are provided, and the conditions have been optimized. When combined with the necessary Duolink® PLA reagents, you will have all you need to successfully perform the PLA experiment and obtain positive results.
The Control kit contains Duolink® PLA Control Slides (Cat. No. DUO82202) - two 8 well glass slides with fixed SK-OV3 Human ovarian cancer cells. The cells were treated with EGF, to induce EGFR and HER2 interaction. The Kit also contains one vial of Anti-HER2 antibody produced in rabbit (HPA001383), and one vial of Monoclonal Anti-EGFR antibody produced in mouse (E3138) – the two primary antibodies you need to detect the two interacting protein
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In the Duolink® PLA Control Kit, the cells and antibodies are provided, and the conditions have been optimized. When combined with the necessary Duolink® PLA reagents, you will have all you need to successfully perform the PLA experiment and obtain positive results.
The Control kit contains Duolink® PLA Control Slides (Cat. No. DUO82202) - two 8 well glass slides with fixed SK-OV3 Human ovarian cancer cells. The cells were treated with EGF, to induce EGFR and HER2 interaction. The Kit also contains one vial of Anti-HER2 antibody produced in rabbit (HPA001383), and one vial of Monoclonal Anti-EGFR antibody produced in mouse (E3138) – the two primary antibodies you need to detect the two interacting protein
Let us do the work for you, learn more about our Custom Service Program to accelerate your Duolink® projects
View full Duolink® product list
Based on proximity ligation assay (PLA), the Duolink® PLA Technology allows for endogenous detection of protein interactions, post-translational modifications, and protein expression levels at the single molecule level in fixed cells.
Duolink® PLA Control Kit - PPI is designed to allows users to gain confidence with the Duolink® PLA technology. This Kit contains glass slides with fixed, treated cells and a specific pair of primary antibodies that will detect interacting proteins and reliably generate PLA signals when used with the complementary Duolink® PLA probes and detection reagents. Analysis is carried out using standard immunofluorescence assay equipment.
Specificity
This kit will enable the detection of Protein-Protein interactions. The SK-OV-3 cells contain HER2 and EGFR, which show increased interaction upon treatment with EGF. When combined with appropriate Duolink® PLA Reagents, the Control Kit will enable the successful detection of increased HER2 and EGFR protein interactions in the EGF treated cells.
Duolink® PLA Control Kit - PPI is designed to allows users to gain confidence with the Duolink® PLA technology. This Kit contains glass slides with fixed, treated cells and a specific pair of primary antibodies that will detect interacting proteins and reliably generate PLA signals when used with the complementary Duolink® PLA probes and detection reagents. Analysis is carried out using standard immunofluorescence assay equipment.
Specificity
This kit will enable the detection of Protein-Protein interactions. The SK-OV-3 cells contain HER2 and EGFR, which show increased interaction upon treatment with EGF. When combined with appropriate Duolink® PLA Reagents, the Control Kit will enable the successful detection of increased HER2 and EGFR protein interactions in the EGF treated cells.
Features and Benefits
- Reliable PLA signal detecting a protein interaction
- Gain confidence with the Duolink® PLA Technology
- Add additional controls to your PLA experiments
- Increased sensitivity due to rolling circle amplification for low abundant targets
- No overexpression or genetic manipulation required
- Relative quantification possible
- Works with any fluorescence microscope and appropriate filters
Other Notes
Other Duolink® reagents needed (30RXN size):
Mouse/Rabbit Duolink® PLA Starter Kit: Red Starter Kit (DUO92101) OR Orange Starter Kit (DUO92102)
OR
PLA Probe pairs (PLUS and MINUS): Anti-Mouse PLUS (DUO92001) and Anti-Rabbit MINUS (DUO92005) or Anti-Mouse MINUS (DUO92004) and Anti-Rabbit PLUS (DUO92002); AND Detection reagents of choice: Orange (DUO92007), Red (DUO92008), FarRed (DUO92013), or Green (DUO92014); AND Wash Buffers (DUO82049) AND Mounting Medium with DAPI (DUO82040).
Mouse/Rabbit Duolink® PLA Starter Kit: Red Starter Kit (DUO92101) OR Orange Starter Kit (DUO92102)
OR
PLA Probe pairs (PLUS and MINUS): Anti-Mouse PLUS (DUO92001) and Anti-Rabbit MINUS (DUO92005) or Anti-Mouse MINUS (DUO92004) and Anti-Rabbit PLUS (DUO92002); AND Detection reagents of choice: Orange (DUO92007), Red (DUO92008), FarRed (DUO92013), or Green (DUO92014); AND Wash Buffers (DUO82049) AND Mounting Medium with DAPI (DUO82040).
This product is comprised of the following:
See datasheet for more information.
- Duolink® PLA Control Slides (DUO82202) - two 8-well chamber slides with EGF treated, pre-fixed SK-OV3 cells.
- One vial (15μL) of Rabbit anti-ErbB2/HER2 antibody (HPA001383)
- One vial (25μL) of Mouse anti-EGFR antibody (E3138)
See datasheet for more information.
Legal Information
Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
RESEARCH USE ONLY. This product is regulated in France when intended to be used for scientific purposes, including for import and export activities (Article L 1211-1 paragraph 2 of the Public Health Code). The purchaser (i.e. enduser) is required to obtain an import authorization from the France Ministry of Research referred in the Article L1245-5-1 II. of Public Health Code. By ordering this product, you are confirming that you have obtained the proper import authorization.
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Articles
包括建议和技巧、常见问题解答和基本故障排除的支持信息。
瞭解 Proximity Ligation Assay 技術如何運作，以及蛋白質-蛋白質互作對照試劑盒如何確認 EGF 誘導的 EGFR-HER2 二聚化的原位檢測。
Support information including tips and tricks, frequently asked questions, and basic troubleshooting.
Learn how Proximity Ligation Assay technology works and how the protein-protein interaction control kit can confirm in situ detection of EGF-induced EGFR-HER2 dimerization.
Protocols
Duolink® PLA reagents enable brightfield detection and quantification of proteins and interactions in tissue samples.
Duolink® PLA 試劑可對組織樣本中的蛋白質和相互作用進行明視場檢測和定量。
Related Content
蛋白質和核酸互作試劑和資源，用於投資蛋白質-RNA、蛋白質-DNA 和蛋白質-蛋白質互作以及相關應用。
Protein and nucleic acid interaction reagents and resources for investing protein-RNA, protein-DNA, and protein-protein interactions and associated applications.
蛋白質和核酸互作試劑和資源，用於投資蛋白質-RNA、蛋白質-DNA 和蛋白質-蛋白質互作以及相關應用。
Instructions
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What is the appropriate course of action if a control is tested with no antibody, only probes, and high background is observed?
1 answer-
Helpful?
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How can I reduce high background staining with Duolink?
1 answer-
Before starting PLA, it is essential to have antibodies that are optimized for IHC with your sample.
Background PLA signals can be distinguished from background autofluorescence. PLA signals are distinct and round, whereas autofluorescence is more diffuse. Background PLA signals are typically related to antibody or PLA probes binding to the sample.
Optimization involves sample blocking, primary antibody titer, sample washing, and ensuring that the sample does not dry. Here are some optimization steps to help reduce background staining:
1. Check the antibody titer to obtain specific and distinct staining. High antibody concentration can lead to many PLA signals in the background outside the cell areas. Optimizing the primary antibody titer can help remove the background staining and allow for distinct staining.
2. Use an appropriate blocking agent. Dilute PLA probes in a buffer containing the blocking agent.
3. Ensure that the sample does not dry out during the Duolink In Situ assay. Use a grease pen to delimit the reaction area, tap off excess solutions gently, and use a humidity chamber.
4. Use Wash Buffers A and B where specified.
5. Wash in at least 70 mL of wash buffers with gentle agitation. A better signal-to-noise ratio is achieved when the proper wash volume is used.
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