Manual / Product Insert

Technical Data Sheet: IHC/ICC Blocking Buffer - High Protein

Version: 14 March 2017 Rev. 10
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Manual / Product Insert

Technical Data Sheet: 20X TBS Wash Buffer for IHC/ICC

Version: 14 March 2017 Rev. 10
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Manual / Product Insert

Technical Data Sheet: BrdU Kit for IHC/ICC Colorimetric

Version: 12/22/2015
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Product FAQ

Can I make custom conjugates with Qdot nanocrystals?

Answer

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.

Answer Id: E12220

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Product FAQ

I am getting very high background with my Qdot streptavidin conjugate. Do you have any suggestions?

Answer

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.

Answer Id: E12243

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Product Literature

Product Line Flyers: Macrophages and monocytes

Manual / Product Insert

A16392_CD115_Rt_x_Ms_PE_mab.pdf

Version: 1.00
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Product Literature

Product Line Flyers: Stem Cells

Product Literature

Handbook: Exploring cancer proliferative signaling pathways (Japanese)

Manual / Product Insert

A16411_CD135_Rt_x_Ms_PECy5_mab.pdf

Version: 1.00
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Manual / Product Insert

A16416_CD115_Rt_x_Ms_APC_mab.pdf

Version: 1.00
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Product Literature

BioProbes 79 Journal of Cell Biology Applications