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View additional product information for Qdot™ 800 Streptavidin Conjugate - FAQs (Q10173MP, Q10171MP)
27 product FAQs found
Here are some suggestions:
Use the Qdot Incubation Buffer (Cat. No. Q20001MP).
The included buffer is formulated specifically for improved signal-to-background ratios in most immunolabeling applications using the Qdot streptavidin conjugates. Alternate buffers may result in more variable staining and, in particular, may increase background staining. However, some specific applications may require other buffer conditions. Please see the protocol "Double-labeling Using Qdot Streptavidin conjugates."
Determine if the sample has a high level of endogenous biotin. Block the sample using an avidin-biotin pre-blocking step.
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 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 contain endogenous biotin, which may contribute to non-specific signal. Endogenous biotin can be blocked with an avidin/biotin blocking kit (Cat. No. E21390).
Grainy staining or clumps of fluorescent material appear 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 in a benchtop centrifuge (Eppendorf 5415) at 5,000 x g for 2 minutes. The material can also be passed over a 0.2 µm spin filter unit before you add it 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 or other salts in the incubation buffer, and may not be easily fixed with filtration. In this case, reduce the overall salt concentration.
Optimize concentration of biotinylated secondary antibodies.
Optimizing specific signal can often be achieved by adjusting the level of biotinylated antibody used instaining. High levels of biotinylated antibody are necessary to obtain specific labeling, but overly high levels will contribute to 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.
Optimize concentration of Qdot streptavidin conjugate.
Just as titration of primary and secondary antibodies is necessary to achieve optimal specific signal in immunolabeling applications, 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.
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Here are some suggestions:
Confirm imaging/detection setup suitability.
Make sure that you are using an appropriate filter set to detect the signal. Please consult Table 1 in the Qdot Biotin User Manual for a list of appropriate and optimal filters.
Check to see that Qdot conjugate is fluorescing using an alternative light source.
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 product does not appear to fluoresce under the long wave UV excitation, please contact Technical Support at techsupport@qdots.com. For a microscope, perform a spot test: place a small droplet (2 to 5 µL) of the quantum dot solution onto a clean slide (no coverslip) and examine under the appropriate filter set at low magnification.
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.
For Qdot streptavidin conjugates, confirm biotinylation of antibody.
Make sure your antibodies 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.
PAP pen ink may quench signal.
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. No. H-4000) from Vector Labs.
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Spinning your ITK Qdot nanocrystals at approximately 3,000 rpm for 3-5 minutes should remove the white precipitate from the supernatant. Use the supernatant immediately.
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The precipitate in the organic ITK Qdot nanocrystals occurs with some frequency. The ITK Qdot nanocrystals sometimes include impurities that show as a white precipitate.
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Blinking is an inherent property of quantum dots; in fact, all single-luminescent molecules blink, including organic dyes. The brightness and photostability of Qdot nanocrystals makes the blinking more visibly apparent. Under higher energy excitation, Qdot nanocrystals blink even faster.
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Appropriate mounting media selection is very important to retain the fluorescence of Qdot nanocrystals. In our studies, Qdot nanocrystals work best with the following mountants:
HistoMount medium (Cat No. 00-8030); best for long term archiving
Cytoseal 60 Mountant
Clarion Mountant
Most polyvinyl alcohol-based mountants (limited storage time, less than weeks)
Water-based mountants (limited storage time, less than week)
Up to 50% glycerol (limited storage time, less than week)
Note: We do not recommend using ProLong mounting media with Qdot nanocrystals as it will quench their fluorescence.
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Freezing will cause the product to aggregate. The Qdot nanocrystals cannot be dispersed into solution after aggregation.
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Once your product undergoes aggregation, it cannot be dispersed back into solution. We recommend purchasing a new product.
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You may occasionally observe a small amount of aggregation of the Qdot nanocrystals during proper storage. To remove any aggregates that may have formed prior to use, we recommend centrifuging the vial at 2,000 x g for 1 min. Pipette only the supernatant and avoid the pellet. In our experience, pelleting any aggregates that may have formed typically results in a loss of less than 10% of the product.
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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.
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The Qdot products contain cadmium and selenium (and tellurium, in the larger particles) 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.
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We have not investigated the toxicity of the Qdot nanocrystals. The materials are provided in a solution which is approximately 2 mM total Cd concentration. 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.
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Qdot 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.
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The Qdot 605 Streptavidin conjugates have stable emission in a number of buffers, across a range of pH. 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 non-aggregated 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 F-68, 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 conjugate at the concentration at which it was shipped, rather than at a higher dilution. Storing materials at working dilution over longer periods of time may result in substantial performance loss. While we have not characterized the stability of the other Qdot conjugates in this variety of buffers, we anticipate similar levels of stability.
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The number of molecules conjugated to one Qdot nanocrystal is based on the ratio of quantum dot:molecule used in the conjugation, the number of available binding sites on the Qdot nanocrystal, and the size of both the Qdot nanocrystal and the molecule of interest. In general, there are 2-3 antibodies, 4-5 biotin molecules, and 6-8 streptavidin molecules per Qdot nanocrystal.
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ITK Qdot nanocrystals use the original formulation of outer polymer provided in the first generation of the Qdot products; except for the Amine-PEG products, the outer polymer does not include PEG. The outer polymer of the standard Qdot nanocrystals includes PEG.
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There are approximately 80-100 functional groups of each Qdot ITK nanocrystal. We use a type of immunosorbent assay to determine the EC50 of each conjugate.
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Yes, you can visualize Qdot nanocrystals using a standard filter; they will excite at any wavelength below their emission. Keep in mind that the lower the excitation value the brighter the Qdot nanocrystal fluorescence output.
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Qdot nanocrystals do not require the use of antifades as they do not photobleach or fade in the same manner as a chemical dye. In our studies, Qdot nanocrystals work best with the following mountants:
- HistoMount medium (Cat No. 00-8030); best for long-term archiving
- Cytoseal 60 Mountant
- Clarion Mountant
- Most polyvinyl alcohol-based mountants (limited storage time, less than a week)
- Water-based mountants (limited storage time, less than a week)
- Up to 50% glycerol (limited storage time, less than a week)
Note: We do not recommend using ProLong or SlowFade mounting media with Qdot nanocrystals.
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Hydrophilic Qdot nanocrystals are stored and shipped in borate buffer pH 8.3-9.0, and organic Qdot nanocrystals are stored and shipped in decane.
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When stored at 4 degrees C, Qdot nanocrystals are stable for approximately 6 months. Qdot nanocrystals should never be frozen due to the possibility of aggregation. The temperature stability of Qdot nanocrystals is summarized below. Please note that fluorescence is not temperature dependent.
<0 degrees C: NEVER freeze Qdot nanocrystals - polymer induces aggregation at freezing temperatures.
>4 degrees C: Core/Shell/Polymer stable at 4 degrees C for ~ 6 months. May be filter sterilized using uncharged filters.
<60 degrees C: Core/Shell/Polymer stable at 60 degrees C (as in in situ hybridization).
<65 degrees C: Core/Shell/Polymer stable at 65 degrees C for only ~1 hour, beyond 1 hour, emission drops off.
<100 degrees C: Core/Shell/Polymer stable up to 100 degrees C brief exposure. OK for 5 minutes at 100 degrees C.
<360 degrees C: Only Core/Shell stable up to 360 degrees C.
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Qdot nanocrystals are most stable at pH 6-9, and marginal stability of Qdot nanocrystals is shown down to a pH 5. Qdot nanocrystals should not be used at pH > 9 due to the possibility of self-aggregation and clumping, and Qdot nanocrystals should not be used pH less than 4 as the polymer and exposed core/shell will begin to dissociate. For more information on Qdot nanocrystals and recommended pH ranges, see pH Ranges for Qdot Nanocrystals (https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/qdot/qdot-reg--nanocrystal0.html)
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You can use Qdot nanocrystals with FRET applications in two scenarios:
- Qdot nanocrystals as donors with fluorescent dyes as acceptors
- Lanthanide (terbium, europium, etc.) as donors with Qdot nanocrystals as acceptors
Note: You cannot perform FRET experiments using Qdot nanocrystals as both donor and acceptor.
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We offer amino (PEG), carboxyl, and streptavidin-functionalized Qdot Innovator's Tool Kit ITK Nanocrystals for the preparation of custom conjugates of proteins or other biomolecules. Amino (PEG)-derivitized forms can be coupled to isothiocyanates and succinimidyl esters or with native carboxylic acids using water-soluble carbodiimides. Carboxyl-derivitized forms can be coupled to amine groups of proteins and modified oligonucleotides. Streptavidin-derivitized forms can be bound with biotinylated conjugates to form stable labeled complexes.
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Qdot nanocrystals and bioconjugates are ideal for experiments requiring long-term photostability or single-excitation, multicolor analysis. Some example applications include:
- Flow cytometry
- Cell and tissue staining
- Cell tracking
- WesternDot western blotting
- In vivo imaging
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Qdot nanocrystals offer many advantages over traditional fluorescent dyes:
- Qdot nanocrystals have a broad excitation range, and they can be excited by any wavelength below their emission peak. The lower the excitation wavelength, the higher the extinction coefficient and Qdot nanocrystal brightness.
- Multicolor detection using Qdot nanocrystals can be done using a single excitation wavelength.
- Qdot nanocrystals exhibit a large Stokes shift.
- Qdot nanocrystals have a narrow emission band.
- Qdot nanocrystals have excellent photostability compared to traditional fluorescent dyes.
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A Qdot nanocrystal is comprises four basic layers. Listed from inner core to outer shell, these are:
1) Core nanocrystal (CdSe or CdSeTe): Determines the color of the Qdot nanocrystal
2) Inorganic shell (ZnS): Improves brightness and stability of the Qdot nanocrystal
3) Organic/polymer coating: Provides water solubility and/or functional groups for conjugation
4) Biomolecule: Covalently attached to the polymer shell and can include antibodies, streptavidin, receptor ligands, or oligonucleotides.
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