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DUO92010

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Duolink® In Situ Probemaker MINUS

Synonym(s):

in situ Proximity Ligation Assay reagent, Protein Protein Interaction Assay reagent

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About This Item

UNSPSC Code:
12352200
NACRES:
NA.32

product line

Duolink®

technique(s)

immunofluorescence: suitable
proximity ligation assay: suitable

suitability

suitable for brightfield
suitable for fluorescence

shipped in

wet ice

storage temp.

−20°C

Related Categories

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. 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 MINUS – one vial with lyophilized activated MINUS 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
GHS07,GHS09

Signal Word

Warning

Hazard Statements

H317,H411

Hazard Classifications

Aquatic Chronic 2 - Skin Sens. 1

Storage Class Code

10 - Combustible liquids

WGK

WGK 3

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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Foivos-Filippos Tsokanos et al.
The EMBO journal, 35(10), 1058-1076 (2016-03-19)
Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates
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
Kan V Lu et al.
Cancer cell, 22(1), 21-35 (2012-07-14)
Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor
Melanie Spears et al.
The Journal of pathology, 227(4), 481-489 (2012-03-21)
The PI3K/Akt signal transduction pathway plays an important role in cancer progression and cell survival. Akt activation is associated with poor outcome in endocrine-treated breast cancer, whereas high levels of cytoplasmic Akt2 are associated with an improved overall survival. Proximity
Ola Söderberg et al.
Nature methods, 3(12), 995-1000 (2006-10-31)
Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses
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