Invitrogen™ SiteClick™ Qdot™ Antibody Labeling Kits

Product Code.	Label or Dye	unitSize
15128735	Qdot 525	Each
15138735	Qdot 655	Each
15136585	Qdot 705	1 set
15148735	Qdot 565	Each
15158735	Qdot 585	Each
15168735	Qdot 625	Each
15188735	Qdot 800	Each
15198735	Qdot 605	Each

Product Code. 15128735 Supplier Invitrogen™ Supplier No. S10449

Description

Achieve precise and reproducible antibody labeling with SiteClick R-PE Antibody Labeling Kit.

Achieve precise and reproducible antibody labeling with SiteClick R-PE Antibody Labeling Kit. This kit targets carbohydrate domains present on all IgG antibodies, regardless of isotype or host species, ensuring site-selective labeling. The labeled antibodies are suitable for flow cytometry, fluorescence imaging, and western blot detection.

Features of the SiteClick R-PE Antibody Labeling Kit include:

• Complete kit—includes everything needed to label 100 μg of IgG antibody using a straightforward, step-by-step protocol
• Site-specific labeling—SiteClick technology attaches labels to the heavy chains of IgG antibodies under mild conditions, preserving antigen-binding domains
• Consistent results—the gentle, site-selective attachment to heavy-chain N-linked glycans ensures reproducibility across sequential labelings and different antibodies, without harsh reduction steps

• Efficient protocol—easy-to-follow steps requiring minimal hands-on time
• High-quality conjugates—produces highly efficient, site-specific, and reproducible antibody conjugates

Unlike traditional amine or thiol labeling, which can affect antigen binding domains or antibody structure, SiteClick labeling technology maintains the integrity of the antibody. This ensures that antigen binding sites remain available for target binding, providing consistent and reproducible labeling. The workflow utilizes copper-free click chemistry to covalently link the DIBO moiety with the azide-modified antibody, ensuring uniform labeling without the need for reaction optimization.

With the SiteClick R-PE Antibody Labeling Kit, you can confidently achieve consistent, high-quality labeling every time.

Specifications

• Kit Contents: Contains everything needed to azido modify and label 100 μg of antibody.
• Product Type: Labeling Kit
• Quantity: 1 kit
• Content And Storage: Store the entire kit at 2 to 6°C upon receipt
• Excitation/Emission: 405/525
• Labeling Method: SiteClick Labeling System
• Labeling Scale: 100 μg
• Shipping Condition: Wet Ice
• Product Line: Qdot, SiteClick
• Label or Dye: Qdot 525
• Color: Green
• Labeling Target: IgG
• Filter Type: GFP, Qdot 525-800
• Flow Cytometer Laser Lines: UV, Violet, Blue

Frequently Asked Questions (FAQs)

I need to label my tissue section with two mouse primary antibodies. Do I have to make direct conjugates of the antibodies to use them?

Check the isotype of the two antibodies to see if they are different. If so, we offer isotype-specific goat anti-mouse secondary antibodies that can be used together with minimal cross-reactivity. If the two mouse antibodies are of the same isotype, then you would need to make direct conjugates, such as with our antibody labeling kits or APEX, and SiteClick kits.

What is the best way to remove white precipitate from my ITK Qdot nanocrystals?

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.

I see a white precipitate in my ITK Qdot nanocrystals; should I be concerned?

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.

Why do my Qdot nanocrystals appear to be blinking?

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.

My Qdot nanocrystals were brightly fluorescent before I mounted my samples; now I'm seeing a loss of fluorescence. Why is this happening?

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.

Why can't I freeze my Qdot nanocrystal solution?

Freezing will cause the product to aggregate. The Qdot nanocrystals cannot be dispersed into solution after aggregation.

My Qdot product is completely aggregated; how do I disperse the aggregates?

Once your product undergoes aggregation, it cannot be dispersed back into solution. We recommend purchasing a new product.

I see a small amount of aggregation in my Qdot product even though I stored it correctly. Why is this happening?

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.

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.

How should I dispose of the Qdot products?

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.

Are the quantum dots toxic?

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.

How many molecules of antibody, streptavidin, and biotin are conjugated to one Qdot nanocrystal?

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.

What is the difference between an ITK Qdot nanocrystal product and a standard Qdot nanocrystal product?

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.

How many functional groups (amino or carboxyl) are loaded onto each Qdot ITK nanocrystal? How do you estimate the number of functional groups?

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.

I don't have a filter optimized for visualizing Qdot nanocrystals. Can I visualize them using a standard filter?

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.

What mounting media should I use with Qdot nanocrystals?

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.

In what solvents are Qdot nanocrystals stable?

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.

What is the temperature range in which Qdot nanocrystals are stable?

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.

What is the pH range in which Qdot nanocrystals are stable?

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)

Can I use Qdot nanocrystals in FRET applications?

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.

Can I make custom conjugates with Qdot nanocrystals?

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.

In which applications can I use Qdot nanocrystals?

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

What advantages do Qdot nanocrystals offer over traditional fluorescent dyes?

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.

What is the basic structure of a Qdot nanocrystal?

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.