Duolink® FAQs

Additional information can be found at sigma.com/duolink.

  1. How long are the oligonucleotide arms?
    Information on the PLA probe sequences is proprietary and cannot be disclosed.

  2. What are the oligonucleotide sequence / chemistry?
    Information on the PLA probe sequences is proprietary and cannot be disclosed.

  3. What does X solution contain?
    Information on the composition of the different reagents is proprietary and cannot be disclosed.

  4. What are the minimum and maximum distances for proximity?
    All the Duolink In Situ products for the in situ PLA detection of protein interactions are designed to detect proximity. When using the secondary antibody approach the theoretical maximum distance between your two target proteins (epitopes) is 40 nm to be able to create a signal. These numbers are based on the average diameter of an antibody being around 6 nm. Duolink can in theory detect epitopes that are within zero nm of each other, as long as the two primary antibodies can bind and there is no steric hindrance.

  5. What is the minimum and maximum number of cells / tissue needed?
    In principle, the minimum number of cells required for a Duolink assay is one cell. The maximum is difficult to establish: if the cell density is very high, it may happen that the antibodies (including the primary antibodies) and reagents have difficulties to reach cells in the center of the sample.
    When it comes to tissue samples, there is no minimum number either. As for the maximum, our R&D department has analyzed tissue slices of up to 30 µm thick with successful results. On the other hand, the success of the experiment will depend both on the tissue sections and its pretreatment (e.g. fixation, permeabilization, epitope retrieval).
    The minimum number of cells will be dependent on your particular application.

  6. In which species have the PLA probes been raised?
    The  secondary antibodies (PLA probes) are derived from farmed donkey (Equus asinus domesticus).

  7. How do I perform a single recognition experiment?
    Performing a single recognition experiment, you will need one primary antibody against your particular target protein and two PLA probes, both directed towards the species of your primary antibody (e.g. if your primary antibody was raised in rabbit, then you will need the PLA probes anti-Rabbit PLUS and anti-Rabbit MINUS). The rest of the Duolink assay is performed as usual.

  8. When can I stop the process?
    There are two steps in the Duolink In Situ protocol that can be run overnight. Please, refer to the protocols in the Duolink In Situ Fluorescence and Brightfield User Manuals in order to follow the following explanations:
    The first step that can be run overnight is incubation of the primary antibody (step 2. Primary antibodies, pages 16-17); depending on the primary antibody, incubation times can vary from half an hour to overnight. Observe that even if the recommended incubation time is short for a particular primary antibody, it may be possible to dilute the antibody and prolong the incubation time to overnight at 4 °C.
    The second step where the protocol can be stopped is different depending upon the detection method:
    - For the fluorescence detection (Duolink In Situ  Fluorescence) protocol (step 6.c Final wash step, page 19): the drying of the slides at room temperature after the final wash step and before mounting the slides can be done overnight. Mounted slides can be stored at +4°C up to four days or sealed and stored at -20 °C for months before analysis. Please note that slides should be stored in the dark!
    - For the HRP detection (Duolink In Situ Brightfield) protocol: after the last dehydration step (step 10.d Dehydration, page 19), slides can be left in fresh xylene or one can proceed with the mounting of the slides (step 11 Preparing for imaging, page 19). Once slides are mounted they need to get dry, usually overnight.

  9. What is the effect of changing incubation times and temperatures?
    The Duolink In Situ protocol has been optimized to obtain results in the shortest time possible without compromising the quality of results. Optimization of incubation times and temperatures has been an important factor in that respect, and therefore, best results are acquired when the protocol is followed with the recommendations given therein.

  10. How long does the PLA signal last / is the signal stable? How should I save my glass slide in order to keep the PLA signals?
    We highly recommend applying our Duolink mounting media both for the fluorescence and the brightfield applications, because they help preserve PLA signals. We have observed that PLA signals can fade when other mounting media is applied, sometimes even immediately after their application on the sample.
    Slides mounted with our non-aqueous (xylene based) and permanent (hard-set) Duolink Brightfield Mounting Medium can be stored at room temperature and PLA signals will last for years.
    Slides mounted with Duolink In Situ Mounting Medium with DAPI can be stored at 4 °C in darkness up to 4 days before they are analyzed with the microscope.
    Unmounted slides can also be saved at -20 °C over the weekend. For long term storage, we recommend applying transparent nail polish around the borders of the cover slip. In this way, the slide can be stored at -20 °C for up to half a year, although normally the signal will fade a little bit with time. If the slide has already been analyzed with the microscope, additional fading may be expected.

  11. How long do the Duolink products last?
    All the reagents in the Detection kit must be kept at -20 °C at all times; this is especially critical for the Ligase and the Polymerase solutions, which need to be kept in a pre-chilled ice-block if taken out from the freezer (an ice-block keeps the enzymes at -20 °C, you cannot achieve that by just having the enzymes on an ice bath). PLA probes, antibody diluents, blocking solutions and mounting medium must be kept at +4 °C.
    We have a 6-month guarantee upon arrival to your laboratory for the Duolink In Situ Probemaker, as long as it is kept at -20 °C. Once the conjugation has taken place, we have noticed that for most conjugated antibodies the shelf life at +4 °C is three to six months. In this case it will be very much dependent on if the antibody requires special preservatives or not.

  12. Can I apply Duolink to my non-fixed in vivo sample?
    Duolink has been optimized for fixed cells and tissues. In a living cell, the enzymes and DNA strands in the Duolink Reagents will probably be deteriorated.

  13. Which antibodies are recommended?
    The primary antibodies should be of IgG-class, specific for the target to be detected and preferably affinity purified. The primary antibodies could be either polyclonal or monoclonal. To maximize your success rate, choose antibodies that are IHC and/or IF classified.

  14. What concentration of my primary antibody should I use?
    If you already have a working assay for IHC or IF, use the same primary antibody concentration to start with. Sometimes it may be necessary to perform a titration of your primary antibody.

  15. My antibodies are from a species other than mouse, rabbit and goat.  Can I still apply Duolink II?
    Yes.  We offer the Duolink In situ Probemaker kit that enables conjugation of the PLA oligonucleotide arms PLUS or MINUS to your antibodies of interest.  In that way, you build your own PLA probes that can later be applied to detect your proteins of interest in combination with Duolink In Situ Reagents.

  16. Both my primary antibodies are from the same species.  Can I still apply Duolink In Situ? 
    Yes.  We offer the Duolink In situ Probemaker kit that enables conjugation of the PLA oligonucleotide arms PLUS or MINUS to your primary antibodies of interest.  In that way, you build your own PLA probes that can later be applied to detect your proteins of interest in combination with Duolink In Situ Reagents.  This allows for the detection of protein homodimers.

  17. When using Duolink Probemaker, can I use a lower concentration of the primary antibody than 1 mg/ml?
    Each kit of Duolink Probemaker contains reagents to conjugate 20 ug of antibody at a concentration of 1 mg/ml.  We strongly recommend to use antibodies at this concentration.  We cannot guarantee a good results with a lower concentration.  If your antibody is at a low concentration, you could concentrate it by ultra-filtration in a centrifuge device.  Note that in this process buffer changes can also be performed by repeated additions of the desired buffer with subsequent centrifugations.

  18. My primary antibodies contains many additives.  What should I do?
    For pre-treatment of the antibody before using Duolink In Situ Probemaker, we refer to standard procedures.
    To change buffer and/or to remove low molecular weight additives like azide or Trehalose, you can either dialyze against PBS or perform gel filtration on a spin column (Sephadex G25) equilibrated with PBS.
    To remove high molecular weight additives such as BSA or gelatin, we recommend purification with Protein A or Protein G.  The antibody is eluted with low pH which might affect antibody activity.  We recommend to immediately add a strong buffer of neutral pH.  There is always a loss of antibody with this purification method.

  19. What dilution of the conjugated antibody should I use?
    The working concentration of the antibody will be dependent mainly on the sensitivity of the antibody, the sample type and the sample pretreatment.  Our general recommendation is to use a starting concentration that gives good results with IF or IHC.  Sometimes, titration may be necessary.

  20. Can I combine a directly conjugated primary antibody and a PLA probe?
    Yes.  It is possible to combine a directly conjugated antibody and a Duolink In Situ PLA probe as long as one of them is PLUS and the other is MINUS and there is no species cross-reactivity (that is, the PLA probe should be directed against a species different from your directly conjugated primary antibody).

  21. Does the Duolink ImageTool work for brightfield?
    Yes.  Duolink ImageTool will detect and quantify PLA signals obtained from your brightfield experiment.