1. Which buffers are compatible with the Qdot Streptavidin Conjugates?
The Qdot 605 Streptavidin Conjugate has stable emission in a number of distinct buffers, across a range of pH conditions. At working concentrations, the quantum yield and colloidal dispersion of these materials have been found to be remarkably stable across pH 6-9 (not investigated outside this range) in Tris, HEPES, phosphate, and borate buffers. The Qdot 605 Streptavidin Conjugate is stable and nonaggregated in buffered NaCl up to 200 mM at working concentrations. Higher salt concentrations may result in microscopic precipitation, but do not appear to cause bulk precipitation of the materials at working dilutions. In addition, a number of surfactants and additives such as Tween™ 20, Triton™-X-100, Pluronic™ F68, NDSB 201, and EDTA, among others have been shown to maintain the fluorescence in 0.05% concentrations. In contrast, gelatin and dextran sulfate were both found to promote aggregation of the Qdot 605 Streptavidin Conjugate at 0.05% concentrations, and should be avoided in labeling applications. In general, we recommend storage of the Qdot nanoparticle conjugates at the concentration at which it is shipped, rather than at a high dilution. Storage of materials at working dilution over longer periods of time may result in substantial performance degradation. While we have not characterized the stability of the other Qdot Streptavidin Conjugates in this variety of buffers, we anticipate similar levels of stability.
2. Which buffers can I use in place of the Qdot Incubation Buffer provided?
Qdot nanoparticle conjugates have higher nonspecific binding in buffers that are not optimized for use with the materials. We have had successful staining results in a variety of buffer conditions, including TBS, PBS, RPMI media, and others, but have found that the performance in the Incubation Buffer is generally predictable and stable.
3. Are the quantum dots toxic?
We have not investigated the toxicity of the Qdot Streptavidin Conjugate. The materials are provided in a solution which is ~2 mM total Cd concentration; however, the CdSe core is encapsulated in a shell of ZnS and the polymer shell, which may prevent dissolution of free Cd. We have demonstrated the utility of these materials in a variety of live-cell in vitro labeling experiments, but do not have systematic data investigating the toxicity of the materials to humans, to animals, or to cells in culture.
4. How should I dispose of the Qdot Streptavidin Conjugate?
The Qdot Streptavidin Conjugate contains cadmium and selenium in an inorganic crystalline form. We can only advise that you dispose of the material in compliance with all applicable local, state, and federal regulations for disposal of these classes of material. For more information on the composition of these materials, consult the Material Safety Data Sheet.
5. Do the quantum dots undergo FRET, or quench when they are in close proximity?
We have not systematically investigated the energy transfer properties of the quantum dots, though the quantum dots may have useful properties as both energy transfer donors and acceptors. We have investigated the fluorescence of Qdot 605 Streptavidin Conjugates that are coupled to each other through a bis-biotin linker, and found that the emission intensity of the materials was unperturbed at any concentration of biotin cross-linker. These results suggest that the interparticle quenching of these Qdot Conjugates is negligible.
6. What are the best filter sets to use with the Qdot Conjugates?
More information on filter selection.
Troubleshooting - No Signal
1. Confirm imaging/detection setup suitability.
Make sure that you are using an appropriate filter set to detect the labeling signals. Please consult Table 1 in the Qdot Biotin User Manual for a list of appropriate and optimal filters or click here.
2. Check to see that Qdot Conjugate is luminescent.
Qdot Conjugates will normally fluoresce brightly under a hand-held ultraviolet lamp (long wave, such as the type used to visualize ethidium bromide on agarose gels). Although we have not seen pronounced loss of fluorescence of these materials under any storage conditions that we have investigated, we have not been able to examine all storage conditions. If the Qdot Streptavidin Conjugates do not appear to fluoresce under the long wave UV excitation, please contact Technical Support for a replacement. (firstname.lastname@example.org 866-440-7368 extension 2, or your local distributor.)
3. Confirm the specificity and titer of primary antibody.
Make sure the antibody will recognize the intended targets. Make sure there is sufficient primary antibody bound to the targets. This verification can be performed by ELISA based capture of the antigen of interest, or by other techniques that can be found in lab manuals such as the Current Protocols in Immunology.
4. Confirm biotinylation of antibody.
Make sure your antibodies (the primary antibodies for two-layer and the secondary antibodies for three-layer detection) are effectively biotinylated. It may be necessary to independently adjust the concentration of both the primary and secondary antibodies used in the assay to obtain optimal signal and minimal background.
5. PAP pen ink may bleach staining signals.
Use an alternate method for isolating target areas on the slide. If your protocol requires the use of a PAP pen, we recommend the ImmEdge Hydrophobic Barrier Pen (Cat. # H-4000) from Vector Labs.
Troubleshooting - High Background
1. Use the Qdot Incubation Buffer.
The included buffer is formulated specifically to achieve improved signal-to-background ratios in most immunohistochemical applications using the Qdot Streptavidin Conjugate. Alternate buffers may result in more variable staining results and in particular may increase the background staining. However, some specific applications may need other buffer conditions. For example, we have observed that using a 5X buffer dilution of the 10X Sigma Blocking Buffer (Sigma Aldrich, St. Louis Mo.Cat # B6429) works better in staining acetone-fixed human epithelial cells. Please see the protocol "Double-labeling Using Qdot Streptavidin Conjugates"
2. Determine if the sample has a high level of endogenous biotin. Block the sample with an avidin-biotin blocking strategy.
If you have used the Qdot Incubation Buffer and still get high nonspecific background, then it may be necessary to check other steps of your procedure. Blocking of the sample with BSA or normal animal serum will generally decrease nonspecific binding of both antibodies and Qdot Streptavidin Conjugates. It is a good practice to dilute your primary and secondary antibodies in the blocking buffer. Some tissues, such as spleen and kidney sections, may have endogenous biotin, which may contribute to non-specific signal. Endogenous biotin can be blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA).
3. Grainy staining or clumps of fluorescent material appears in the background.
Occasionally the BSA within the Qdot Incubation Buffer shows slight aggregation over time. It is necessary to remove this aggregate prior to labeling the sample with the Qdot Streptavidin Conjugate. Spin down the incubation mixture before addition to the sample. This can be accomplished by spinning the samples on a benchtop centrifuge (Eppendorf 5415) at 5000 xg for 2 minutes. The materials can also be passed over a 0.2 µm spin filter unit prior to addition to the sample for staining to remove microscopic precipitates. If you are using a buffer that is different than the Qdot Incubation Buffer, this behavior can often be attributed to higher levels of NaCl in the incubation buffer, and may not be easily fixed with filtration. Reduce the overall salt concentration to reduce this problem.
4. Optimize concentration of biotinylated secondary antibodies.
Adjusting the level of biotinylated antibody for the staining can often be used to optimize the specific signal. High levels of biotinylated antibody are necessary to obtain the specific labeling, but overly high levels will contribute to the nonspecific binding of the antibody to the sample. Nonspecifically bound biotinylated antibody will bind to the Qdot Streptavidin Conjugate, resulting in higher staining of the background samples.
5. Optimize concentration of Qdot Streptavidin Conjugate.
Just as titration of primary and secondary antibodies are necessary to achieve optimal specific signal in immunohistochemical applications, adjustment of the level of the final probe should be optimized for each conjugate. In general, concentrations at or slightly below saturation should have the optimal signal-to-background ratio, while concentrations substantially higher than saturation will compromise the assay with higher background levels.