Alexa Fluor® Secondaries - Antibody Validation, Quality and Specificity

The following article presents information on how to get the best results in immunofluorescence with the fluorescent secondary antibodies conjugated to Alexa Fluor® and optimized for cell staining, flow cytometry, and fluorescent western blot.

Validation

The products have been thoroughly tested in applications, such as cell imaging, flow cytometry and fluorescent western blotting. Therefore, effective performance is guaranteed for every single product due to the stringent QC procedures.

Quality

The optimization of ratio of dye molecules per secondary antibody ensures both the minimization of photobleaching and bright staining. Moreover, there is a column separation step to remove free dye in each and every secondary conjugate. The removal of free dye secures minimal background staining.

Specificity

Alexa Fluor® conjugated secondary antibodies against particular IgG subclasses, such as IgG2a and IgG2b, are now available. These help to detect a number of targets in different species, to which primary antibodies are not available.

The catalog of mouse monoclonal primary antibodies is varied and it has been developed over the course of many years. The detection of subclass-specific mouse monoclonal antibodies in a reliable way is also achievable and it makes the multiplex experiments far more flexible.

Customer Review

‘The spectral location in the red made this perfect for a double label with the other fluorophore in the blue... It has stood up well to storage in the refrigerator and gives a good signal after six months.

Example Data

Cell Imaging

Image: courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical school.

Image: courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical school.

Sample

Embryoid bodies (EBs) acquired upon differentiation of mouse embryonic stem cells.

Green Staining

Primary antibody rabbit anti-laminin. Secondary antibody goat anti-rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081).

Red Staining

Primary antibody mouse anti-disabled 2. Secondary antibody goat anti-mouse IgG - H&L (Alexa Fluor® 594) pre-adsorbed (ab150120).

Blue Staining

Nuclei counter stained with DAPI.

Fluorescent Western Blot

Sample

HeLa cell extract - lanes 1 to 5, help to decrease the total amount of cell protein, 25 µg, 20 µg, 15 µg, 10 µg and 7 µg.

Red Staining

Alpha tubulin (primary ab18251 + anti-rabbit Alexa Fluor® 790, ab175773).

Green Staining

GAPDH (primary ab83956) + anti-chicken Alexa Fluor® 680, ab175787).

Flow Cytometry

Summary

Overlay histogram shows the specificity of the rat anti-mouse IgG3 gamma chain subclass. Alexa Fluor® 647 (ab172329) reacts with IgG3 (red) primary mouse, but does not react with other mouse IgG subtypes, such as IgG1 (black), IgG2a (green), IgG2b (pink) and IgM (light blue).

Alexa Fluor® Secondaries

Alexa Fluor® Color in vial* Absorption Max (nm) Emission Max (nm) Emission Color** Matching
Dyes
405 Colorless 402 421 Blue Cascade Blue
488 Yellow 495 519 Green Cy2, FITC (fluorescein)
555 Pink 555 565 Orange Cy3, TRITC (Rhodamine)
568 Violet 578 603 Orange/ Red Rhodamine Red
594 Purple 590 617 Red Texas Red
647 Blue 650 668 Far Red APC, Cy5
680 Blue 679 702 Near-IR*** Cy5.5, IR680
750 Cyan 749 775 Near-IR*** Cy7
790 Cyan 784 814 Near-IR*** IR800

* The way the solution appears in the vial.
** The usual emission color seen through a conventional fluorescence microscope with appropriate filters.
*** The light emitted by near-IF fluorescent dyes is not accessible to the human vision as the human eye can only process light of up to 650 nm.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Apr 19, 2019 at 3:42 AM

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