Invitrogen™ Wheat Germ Agglutinin (WGA)

Catalog No.	Conjugate
Q12021MP	Qdot 655
W11263	Alexa Fluor 350
W7024	Alexa Fluor 350, Oregon Green 488, Tetramethylrhodamine, Texas Red
W11261	Alexa Fluor 488
W32464	Alexa Fluor 555
W11262	Alexa Fluor 594
W21404	Alexa Fluor 633
W32466	Alexa Fluor 647
W32465	Alexa Fluor 680
W56132	Alexa Fluor Plus 405
W56133	Alexa Fluor Plus 568
W56134	Alexa Fluor Plus 770
W834	Fluorescein
W6748	Oregon Green 488
W849	Tetramethylrhodamine
W21405	Texas Red-X

Catalog No. Q12021MP Supplier Invitrogen™ Supplier No. Q12021MP

Description

Thermo Fisher Scientific offers bright conjugates of wheat germ agglutinin (WGA) and Alexa Fluor, Alexa Fluor Plus, and other dyes. Fluorescent WGA conjugates bind to carbohydrates and are used for various cell biology applications such as plasma membrane labeling and cell painting assays.

Thermo Fisher Scientific offers a broad selection of fluorescent wheat germ agglutinin conjugates. These lectins can bind to carbohydrates and are available conjugated to Alexa Fluor™, Alexa Fluor™ Plus, and other fluorescent dyes. Fluorescent wheat germ agglutinin conjugates are valuable tools in molecular and cell biology research, enabling researchers to label the plasma membrane in fluorescence imaging and cell painting assays, and study and analyze glycosylation patterns and glycan-mediated processes in cells and tissues.

Thermo Fisher Scientific offers bright conjugates of wheat germ agglutinin (WGA) with Alexa Fluor, Alexa Fluor Plus, and other dyes. WGA is a cell impermeant stain that selectively binds to N‐acetylglucosamine and N‐acetylneuraminic acid (sialic acid) residues, which are often found on cell membranes. Fluorescent WGA conjugates provide selective labeling of the plasma membrane with minimal background in many cell types that is retained after formaldehyde fixation and permeabilization with Triton X-100.

These fluorescent lectin conjugates can also be used to label fixed cells; however, to avoid labeling intracellular components, formaldehyde-fixed cells should not be permeabilized before labeling. Fluorescent WGA conjugates are used as plasma membrane stains along with other cellular markers in cell painting assays to provide a phenotypic readout of cell health or cytotoxicity. The Wheat Germ Agglutinin, Alexa Fluor 555 Conjugate is included in the Image-iT Cell Painting Kit (Cat. Nos. I65000 and I65500).

WGA conjugates are also used as retrograde tracers for neuronal tracing experiments and have been shown to cross synapses. These fluorescent lectins are applicable in microbiology studies to label yeast bud scars, the cell membrane of gram-positive but not gram-negative bacteria, and chitin in fungal cell walls. In solution, WGA exists as a heterodimer with a molecular weight of approximately 38,000 Daltons and is normally cationic under physiological conditions. Our WGA conjugates have been used in variety of applications, including immunofluorescence (IF), immunohistochemistry (IHC), flow cytometry (FC), and a wide range of chemical, biochemical and immunological assays.

Thermo Fisher Scientific offers a broad selection of fluorescent wheat germ agglutinin conjugates with options covering the entire wavelength range. The Wheat Germ Agglutinin Sampler Kit (Cat. No. W7024) includes introductory samples of four fluorescent WGAs: Alexa Fluor 350, Oregon Green 488, tetramethylrhodamine, and Texas Red-X conjugates. The red-fluorescent Alexa Fluor 594 wheat germ agglutinin (WGA) conjugate is also included in the Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit (Cat. No. I34406). The Texas Red-X WGA conjugate can be purchased in the ViaGram Red+ Bacterial Gram Stain and Viability Kit (Cat. No. V7023) to differentiate gram-positive and gram-negative bacteria.

Wheat germ agglutinin (WGA) conjugates fluorescence excitation/emission
W11261 WGA, Alexa Fluor 488 conjugate: 495 nm/519 nm
W11263 WGA, Alexa Fluor 350 conjugate: 346 nm/442 nm
W32464 WGA, Alexa Fluor 555 conjugate: 555 nm/580 nm
W11262 WGA, Alexa Fluor 594 conjugate: 590 nm/617 nm
W21404 WGA, Alexa Fluor 633 conjugate: 632 nm/647 nm
W32466 WGA, Alexa Fluor 647 conjugate: 650 nm/665 nm
W32465 WGA, Alexa Fluor 680 conjugate: 679 nm/702 nm
W56132 WGA, Alexa Fluor Plus 405 conjugate: 408 nm/450 nm
W56133 WGA, Alexa Fluor Plus 568 conjugate: 562 nm/583 nm
W56134 WGA, Alexa Fluor Plus 770 conjugate: 770 nm/797 nm
W834 WGA, fluorescein conjugate: 494 nm/518 nm
W6748 WGA, Oregon Green 488 conjugate: 496 nm/524 nm
W849 WGA, tetramethylrhodamine conjugate: 555 nm/580 nm
W21405 WGA, Texas Red-X conjugate: 595 nm/615 nm

Specifications

• Concentration: 1 μM
• For Use With (Application): Flow Cytometry, Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Cell Painting
• Quantity: 200 μL
• Source: Wheat germ
• Common Name: Wheat Germ Agglutinin, Qdot™ 655 Conjugate
• Conjugate: Qdot 655
• Species: Wheat
• Recombinant: Native
• Protein Tag: None
• Content And Storage: Store in refrigerator (2–8°C).
  
  
• Shipping Condition: Room Temperature
• Expression System: Wheat germ
• Protein Family: Lectins
• Protein Form: Heterodimer
• Form: Solution
• Protein Subtype: Agglutinin
• Product Line: Qdot
• Purity or Quality Grade: See Certificate of Analysis
• Protein: Fluorescent lectins
• Ligand Type: N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues

Frequently Asked Questions (FAQs)

My cells are very sensitive and need to be kept in media as much as possible. Is it possible to label the plasma membrane with fluorescent wheat germ agglutinin (WGA) in media instead of buffer?

Yes. Although labeling in buffer (such as Hank's Buffered Saline Solution) is slightly better for brightness and lower non-cell background, media can be used. Do a concentration range to dertermine optimal conditions, since the WGA may potentially bind media components to some extent, slightly decreasing your specific labeling intensity.

How do I know which tracer to choose for my experiment?

Factors to consider are size of tracer, method of delivery (injection, direct application to tissue, etc.), and if the tracer needs to be fixable. Here are some links to details about the various classes of neuronal tracers we offer and how to choose between them:

Neuronal Tracing (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html)
Choosing a Tracer (https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorescent-tracers-of-cell-morphology-and-fluid-flow/choosing-a-tracer.html)
Imaging Analysis (http://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/bioprobes-50-journal.pdf)

I want to label the plasma membrane of my cells, but there are several dyes to choose from. Which one should I use?

For live-cell imaging, the CellVue and CellMask Plasma Membrane Stains are the most uniform and the slowest to be endocytosed. However, they are not the best choice if you wish to fix and permeabilize your cells, such as for antibody labeling. Wheat germ agglutinin (WGA) conjugates are also able to label live cells, or can label already formaldehyde-fixed cells. They can survive subsequent permeabilization with detergents, such as Triton X-100. If cells are already permeabilized, WGA will label internal structures as well. Thus, only an antibody against a plasma membrane protein can be used if cells are already permeabilized. Lipophilic cyanine dyes, such as DiI, will label all cell membranes in live cells, not just plasma membranes, if left on live cells for extended periods. Following page will help you choose (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-structure/plasma-membrane.html).

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.

For Research Use Only. Not for use in diagnostic procedures.