All tags Secondary antibodies Validated Alexa Fluor® secondaries for guaranteed performance

Validated Alexa Fluor® secondaries for guaranteed performance

Validation

Products have been extensively tested within our own facilities in applications such as cell imaging, flow cytometry and/or fluorescent western blotting. Robust QC procedures guarantee high performance for each individual product.

Quality

The ratio of dye molecules per secondary antibody have been optimized to minimize photobleaching and ensure bright staining. Furthermore, each secondary conjugate undergoes a column separation step to remove free dye thus ensuring minimal background staining.

Specificity

We now offer Alexa Fluor® conjugated secondary antibodies against specific IgG subclasses (e.g. IgG2a and IgG2b) for the detection of multiple targets to which primary antibodies raised in different species are not available.

A large catalogue of mouse monoclonal primary antibodies has been developed over the years. The possibility to detect subclass-specific mouse monoclonal antibodies in a reliable way adds enormous flexibility to your multiplex experiments.

Customer Abreview

"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 6 months."

Example data

Cell imaging

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

Sample
Embryoid bodies (EBs) obtained 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, decreasing amounts of total cell protein, 25µg, 20µg, 15µg, 10µg and 7µg.

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

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

Flow cytometry

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

ColorPrimary AbSecondary Ab (ab172329)
Dark blueNoNo
Black

ab8989 - mouse monoclonal (isotype IgG1) to Lamin B1

Yes
Greenab49105 - mouse monoclonal (isotype IgG2a) to alpha 1 cateninYes
Pinkab16965 - mouse monoclonal (isotype IgG2b) to thioredoxinYes
Redab60602 - mouse monoclonal (isotype IgG3) to NME2Yes
Light Blueab55602 - mouse monoclonal (isotype IgM) to PKM2Yes
BrownNoYes


Alexa Fluor® secondaries

Alexa Fluor®Color in vial*Absorption Max (nm)Emission Max (nm)Emission Color**Matching Dyes
405Colorless402421BlueCascade Blue
488Yellow495519GreenCy2, FITC (fluorescein)
555Pink555565OrangeCy3, TRITC (Rhodamine)
568Violet578603Orange/ RedRhodamine Red
594Purple590617RedTexas Red
647Blue650668Far RedAPC, Cy5
680Blue679702Near-IR***Cy5.5, IR680
750Cyan749775Near-IR***Cy7
790Cyan784814Near-IR***IR800

* Appearance of the solution in the vial

** Typical emission color seen through a conventional fluorescence microscope with appropriate filters

*** It is not possible to view the light emitted by near-IF fluorescent dyes since human vision is insensitive to light beyond 650 nm approximately.


Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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