Quick Quantum Dot Update

In a previous post, I explained problems encountered with a quantum dot (Qdot)-conjugated secondary antibody. Since then, I have received a replacement Qdot-conjugated secondary antibody (Q22085: Donkey anti-Mouse IgG (H+L) Secondary Antibody, Qdot 625 conjugate) and have had success with labelling tubulin of HeLa cells with Qdots, Hurrah! This is clear from microscope images, where tubulin has been dual-labelled with both an organic dye (Alexa Fluor 488) and the Qdot-conjugated secondary antibody (Figure 1).

 Dual_Labelling_Tubulin_Blog

Figure 1. Dual-Labelling of Tubulin. Fixed HeLa cells (A) were immunostained with an anti-tubulin primary antibody, an anti-mouse QD-conjugated secondary antibody (B), an anti-mouse Alexa Fluor 488 antibody (C) to produce a dual-labelled image (D). Nuclei (E) were also stained with Hoechst 33342.

Although, the Qdot-conjugated secondary antibody can successfully label tubulin with little or no aspecific staining, it does not appear to label any other protein I have tried so far; including the nuclear protein SC35 or proteins more accessible to Qdots such as the foci adhesion protein talin (Figure 2).

Talin_dual_labelled_with_Alexa_488_and_QD_Blog

Figure 2. Dual-Labelling of Talin. Fixed HeLa cells (A) were immunostained with an anti-talin primary antibody, an anti-mouse QD-conjugated secondary antibody (B), an anti-mouse Alexa Fluor 488 antibody (C) to produce a dual-labelled image (D). Nuclei (E) were also stained with Hoechst 33342.

Many optimisations were tried and the primary antibodies have consistently shown to work well with several organic dyes, so it puzzles me why this Qdot-conjugated secondary antibody only seems to work for tubulin. On a more positive note, Qdot labelled tubulin images are now being post-processed using Super-resolution Optical Fluctuation Imaging (SOFI) to enhance resolution.

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9 thoughts on “Quick Quantum Dot Update

  1. If you leave out the primary does the Q-dot no longer label? If so then there is specificity. If the secondary picks out tubulin without the primary, then the secondary is ‘specifically non-specific’.

    Is the secondary on the Q-dot isotype specific? Your other primaries may be wrong isotope. Rare, as secondaries are usually pretty generic.

    Have you tested the Q-dot secondary in a controlled in vitro system?

    There may be an access effect with the antibody passivated Q-dot or passivated Q-dot-antibody conjugate. Have you tried something very accessible – on the apical surface of the cells?

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    • I have done a secondary antibody control with no primary antibody under the same conditions. With both the organic dye and the Qdot-conjugated secondary antibody there was very little or no aspecific staining (low signal) and no tubulin labelling.

      Both the Qdot-conjugated secondary antibody and the organic dye Alexa Fluor 488 have the isotype IgG (H+L). All the primary antibodies I have used have the isotype IgG1.

      The Qdot-conjugated secondary antibody was tested in vitro on HeLa cells, which were grown on glass coverslips and then fixed.

      I have not yet tried any primary antibodies that specifically target epitopes, which are on the surface of cells. I assumed that even though nuclear proteins may be difficult for Qdot-conjugates to access, talin should be easily accessible with sufficient permeabilisation; so surface proteins, such as integrin are something interesting to try. Thank you for your suggestion, much appreciated.

      One explanation suggested for the Qdot-conjugated antibody binding to tubulin rather than the other proteins tried was that tubulin is abundant in cells, so there are many more antigens along the structure.

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  2. In response to Steve Royle @clathrin, who asked on twitter:

    “can she list out the abs used (+ species) and rough protocol?”

    Primary antibodies used:

    Sigma-Aldrich T3287-.2ML, Anti-Talin-1 monoclonal antibody produced in mouse (clone 8d4), species reactivity: Frog, mouse, rat, human, and chicken

    Sigma-Aldrich T5201-200UL, Anti-Beta-Tubulin antibody produced in mouse (clone TUB 2.1), species reactivity: chicken, human, frog, wheat, plant, rabbit, bovine, mouse, hamster, moth, rat, sea urchin

    Abcam ab11826, Anti-SC35 nuclear speckle marker, Mouse monoclonal, Species reactivity: Mouse, Rat, Human, Xenopus laevis, Fruit fly (Drosophila melanogaster), Rhesus Monkey, Newt

    Secondary antibodies used:

    ThermoFisher Scientific, Donkey anti-Mouse IgG (H+L) Secondary Antibody Qdot 625 conjugate, Polyclonal (Q22085)

    ThermoFisher Scientific, Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate, Polyclonal (A-11001)

    Protocol (based upon the protocol suggested on the ThermoFisher Scientific website):

    1) Briefly washed cells in 1x PBS (37°C)
    2) Fixed with 4% PFA (10 min)
    3) Washed 3x with 1x PBS (5 min)
    4) Incubated with 0.25% Triton X-100 in 1x PBS (15 min)*
    5) Incubated with 6% BSA in PBS (60 min)
    6) Incubated with primary antibody diluted in 6% BSA on parafilm in a humidified chamber at 4°C overnight**
    7) Washed 3x with 1x PBS (5 min)
    8) Incubated with secondary antibody (mixture of Qdot-conjugated secondary antibody and Alexa Fluor 488) diluted in 6% BSA on parafilm in a humidified chamber at room temperature (60 min)
    9) Washed 3x with 1x PBS (10 min)
    10) Added 500 uL Hoechst (10 min)
    11) Washed 3x with 1x PBS (10 min)
    12) Mounted with Dako Fluorescent mounting media
    13) Stored at 4°C

    *Variable permeabilisation has been tried: 0.1-0.5% at 15-60 min
    **As well as overnight incubation, 60 min incubation has been tried both at room temperature and 4°C

    I hope this clarification helps 🙂

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    • It does. It disproves the two thoughts I had. First was that the primaries were not matched (I think you said on the other page that the tubulin was a rabbit Ab), but they are. Second was that the secondary incubations, if sequential, could reduce detection of primary. However, you are incubating together so that’s ruled out too.

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      • I am not sure that incubating together rule out the problems you expect with sequencial incubation. An antibody with a dye is usually one antibody with 2-5 dye molecules on it. An antibody-QD conjugate is usually a QD with a few antibodies on it. That means that QD conjugates are both bigger in size and diffuse slower. Therefore effectively they can reach the primaries after (or get depleted from the immidiate area of the primary earlier).

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  3. Here is a potential though imaginative explanation: the secondary Ab does not recognize a single primary, but a pair of primary when they are in a certain configuration which is the one present when the primary are assembled onto tubulin. While this would explain the data, it seems not plausible in terms of selection of the secondary Ab. An alternative, related, hypothesis would be that the secondary affinity is weak but that is enough in the presence of multiple close sites (a sort of avidity effect) as exist in the case of tubulin fibers.

    Now, fire… (and then suggest other ideas?)

    Thanks for your help!

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    • Maybe… I don’t have experience with the other primaries, but tubulin abs (like TUB2.1) bind very well at low low concentrations. It could just be that you have many more primaries bound in this case, not necessarily their spatial arrangement, i.e. the Qdot secondary is not as sensitive and this matters for Talin or nuclear speckle protein, but not for tubulin.
      I don’t like Dave’s suggestion (sorry), this is taking you further away from what you want to do, which is presumably to label the structures in cells with QDots. If they don’t have to be QDots, we have had good experiences recently with FluoroNanoGold conjugated secondaries.

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  4. One cannot exclude this explanation.

    I think it would be useful to try this with a system where you have control over the concentration of the antigen. You could buy a mouse anti FGF2 and play in vitro and on the outside of the cell with a reagent we have buckets of (FGF2) and whose concentration at the cell surface you can control. This would also allow you to use a streptvidin-QD, as we can easily biotinylate FGF2 in two different ways.

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