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DUO92009

Sigma-Aldrich

Duolink® In Situ Probemaker PLUS

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Synonym(s):
in situ Proximity Ligation Assay reagent, Protein Protein Interaction Assay reagent
NACRES:
NA.32

product line

Duolink®

Quality Level

technique(s)

immunofluorescence: suitable
proximity ligation assay: suitable

suitability

suitable for brightfield
suitable for fluorescence

shipped in

wet ice

storage temp.

−20°C

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DUO92010DUO82040DUO92001
technique(s)

immunofluorescence: suitable, proximity ligation assay: suitable

technique(s)

immunofluorescence: suitable, proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

technique(s)

immunofluorescence: suitable, proximity ligation assay: suitable

suitability

suitable for brightfield, suitable for fluorescence

suitability

suitable for brightfield, suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for brightfield, suitable for fluorescence

shipped in

wet ice

shipped in

wet ice

shipped in

wet ice

shipped in

wet ice

storage temp.

−20°C

storage temp.

−20°C

storage temp.

2-8°C

storage temp.

2-8°C

Application

Test your primary antibodies (IgG-class, mono- or polyclonal) in a standard immunofluorescence (IF), immunohistochemistry (IHC) or immunocytochemistry (ICC) assay to determine the optimal fixation, blocking, and titer conditions. Duolink® in situ reagents are suitable for use on fixed cells, cytospin cells, cells grown on slide, formalin-fixed, paraffin embedded (FFPE), or tissue (fresh or frozen). No minimum number of cells is required.

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To perform a complete Duolink® PLA in situ experiment you will need two primary antibodies (PLA, IHC, ICC or IF validated) that recognize two target epitopes. Other necessary reagents include a pair of PLA probes from different species (one PLUS and one MINUS), detection reagents, wash buffers, and mounting medium. Note that the primary antibodies must come from the same species as the Duolink® PLA probes. Analysis is carried out using standard immunofluorescence assay equipment.HRP is also available for brightfield detection.

Specificity
Duolink® In Situ Probemaker extends your possibilities even further by allowing you to create your own PLA probes to meet specific assay requirements.

  • Study homodimers
  • Study protein-protein interactions with two primary antibodies derived from the same species
  • Study protein-protein interactions with primary antibodies from any species

Using Duolink® In Situ Probemaker PLUS and MINUS, you simply conjugate the PLUS and MINUS oligo arms directly to your antibodies in a quick and convenient procedure. As well as providing unique capabilities, this also means there is no longer any limitation as to which species your primary antibodies have to be derived from in order perform a Duolink® In Situ assay.

Application Note
The antibody to be conjugated must have a concentration of 1 mg/ml. Do not use volumes larger than 20 ml for conjugation. The antibody must be in an amine free buffer, ideally PBS. The buffer should be carrier free but may contain up to 0.1% BSA, 5% trehalose, and 0.02% sodium azide.
  • Study homodimers by using one monoclonal antibody split into two halves. Label one with the PLUS oligo and the other with the MINUS oligo.
  • Use antibodies from same species: study protein-protein interactions, protein modifications, or single proteins, using two primary antibodies derived from the same species. Label one of the antibodies with the PLUS oligo and the other with the MINUS oligo
  • Use antibodies from any species: study protein-protein interactions, protein modifications, or single proteins, using one or both primary antibodies derived from species other than mouse, rabbit or goat. Label one of your secondary antibodies with the PLUS oligo and the other with the MINUS oligo. Or, combine one labeled secondary antibody with a standard secondary Duolink®In Situ PLA probe.

Features and Benefits

  • No overexpression or genetic manipulation required
  • High specificity (fewer false positives)
  • Single molecule sensitivity due to rolling circle amplification
  • Relative quantification possible
  • No special equipment needed
  • Quicker and simpler than FRET
  • Increased accuracy compared to co-IP
  • Publication-ready results

Components

This product is comprised of the following:
  • Duolink® In Situ oligonucleotide PLUS – one vial with lyophilized activated PLUS oligonucleotide for one conjugation of 20 μg antibody
  • Conjugation buffer – buffer for conjugation reaction
  • Stop reagent – stops conjugation reaction
  • Storage solution – buffer for preserving prepared PLA probe (conjugated antibody)
  • 20x Assay reagent – reagent to be added to experimenter optimized antibody diluent
  • Blocking solution – blocks sample prior to staining with Duolink® In Situ
  • PLA probe diluent – buffer for diluting PLA probe (conjugated antibody) to final assay concentration
See datasheet for more information.

Other Notes

Duolink®proximity ligation assay(PLA®) allows for endogenous detection of protein interactions, post translational modifications, and protein expression levels at the single molecule level in fixed cells and tissue samples.

Follow the Duolink®In Situ Probemaker Protocol to use the Duolink®In Situ Probemaker PLUS and MINUS.This product can be applied to both the Duolink® In Situ Fluorescence Protocol and the Duolink® In Situ Brightfield Protocol depending on the detection reagents used.For more information, see Create Your Own PLA® Probes.

Visit our Duolink® PLA Resource Center for information on how to run a Duolink® experiment, applications, troubleshooting, and more.

Legal Information

Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany

Pictograms

Exclamation markEnvironment

Signal Word

Warning

Hazard Statements

Hazard Classifications

Aquatic Chronic 2 - Skin Sens. 1

Storage Class Code

10 - Combustible liquids


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Camille Hallez et al.
Nature microbiology, 4(7), 1196-1207 (2019-04-03)
Numerous human APOBEC3 cytidine deaminases have proven to be, inter alia, host cell restriction factors for retroviruses and hepadnaviruses. Although they can bind to genomic RNA and become encapsidated, they are only catalytically active on single-stranded DNA. As there are
Roberto Di Maio et al.
Science translational medicine, 10(451) (2018-07-27)
Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately
Annabel Christ et al.
Developmental cell, 22(2), 268-278 (2012-02-22)
Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM)
Lei Zhu et al.
The Prostate, 73(2), 219-226 (2012-07-19)
PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including α(1) -antichymotrypsin (ACT). The proportion of the PSA-ACT complex is
Charles Lu et al.
PloS one, 7(4), e34833-e34833 (2012-05-05)
Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of-function genetic abnormalities. We

Articles

Things to consider for preparation, setup and execution of the Duolink® assay protocol

Support information including tips and tricks, frequently asked questions, and basic troubleshooting.

Protocols

Duolink® kits use in situ PLA®, a proximity ligation assay technology, to accurately and objectively quantify individual proteins, and their interactions and modifications in unmodified cells and tissue.

Related Content

Applications to detect, quantify and visualize protein-protein interactions, post-translational modifications and low expression protein detection using proximity ligation assay

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