Alexa Fluor® conjugated secondary antibodies - FAQs

by Javier Conde Vancells

To make it easier for customers to use our Alexa Fluor® secondary antibodies I interviewed Brendan Collins (scientific support representative) to discuss your commonly asked questions. Together we wrote the following FAQs section.

At what temperature should I store the antibody?

Storage at 4°C should not exceed 1 or 2 weeks. Upon arrival, you should briefly spin the antibody tube to pull-down the solution and aliquot the secondary antibody solution. Aliquots should be stored at -20°C or -80°C.

Why should I aliquot the antibody?

Aliquoting minimizes the possibility of contamination and prevents the degradation of the antibody due to continuous freeze/thaw cycles. Avoid preparing aliquots smaller than 10 µl as stock concentrations may be affected due to evaporation and adsorption of the antibody onto the surface of the vial.

What type of tubes do you recommend for storage?

Aliquots should be stored in dark low-protein-binding tubes. Prolonged exposure to light would compromise the antibody activity due to photo-bleaching of the fluorophores.

Can I dilute the antibody for storage?

Antibodies may be preferably diluted in the same buffer in which they are shipped (0.02% sodium azide, 1% BSA, 30% glycerol, PBS). Alternatively, you may dilute the antibody in PBS. However, we recommend storing antibodies in their concentrated form as extensive dilution may affect their stability.

What blocking solution should I use with the antibody?

The datasheet indicates the most appropriate blocking solution for your experiment. The blocking solution should be made fresh before each use. Common blocking agents for specific applications are listed below. 

ApplicationBlocking Agent*
Western blot (WB)5% BSA or 5% non-fat milk
Immunofluorescence/ Immunocytochemistry (IF/ICC)10% serum from the species in which the secondary antibody was raised in. Alternatively, 1% BSA or 1% gelatin

* Blocking agents should be fully dissolved before adding the antibody. Undissolved blocking agents may result in artifacts being generated during your experiments. 

In which solution should I dilute the antibody for sample incubation?

Diluting your antibody in blocking buffer for sample incubation may help minimize background. The sample may also be incubated with an antibody solution lacking a blocking agent such as TBST (Tris Buffered Saline with Tween® 20). The results may vary, but if background is not an issue we recommend diluting the antibody in buffer with low concentrations of blocking agent.

How long should I incubate my sample with the antibody?

Incubation times will mostly depend on the expression of your protein of interest. Generally, WB and IF/ICC will only require a 1 hour incubation for effective detection. 

May exposure to light during sample incubation affect my results?

Samples should be incubated in the dark when using Alexa Fluor® conjugated secondary antibodies. Extensive exposure to light could result in photobleaching of the dye and thus affect secondary antibody conjugate performance.

At what temperature should I incubate my sample with the secondary antibody solution?

The temperature at which your sample should be incubated with the Alexa Fluor® conjugated secondary antibody will be usually dictated by the incubation time.

Incubation TimeTemperature
Short (e.g. 1 hour)Room temperature
Long (e.g. overnight)4°C

What is the optimal dilution for my antibody?

Dilution of the antibody for detection may vary depending on the application, the experimental conditions, and your specific target. A good starting point is to dilute the secondary antibody ten times more than your primary. However, a titration curve using serial dilutions of the secondary antibody should be made to determine the optimal dilution. Titration should mimic the conditions of the intended experiment.

How can I minimize background?

The intense fluorescence of Alexa Fluor® conjugated secondary antibodies provides greater signal amplification than other similar dyes. Secondary antibodies are usually diluted in blocking buffer during sample incubation to minimize non-specific binding. Working at an optimal dilution of your secondary antibody will also help minimize background. Make sure you wash extensively your sample after incubation with the antibody. You should also be aware that long incubation times such as overnight may result in increased background.

Can I re-use the secondary antibody solution?

For optimal results, we do not recommend re-using Alexa Fluor® conjugated secondary antibodies. If re-using the secondary antibody solution, fresh antibody should be added as the effective concentration is lowered after each use.

What can I use as a negative control?

To test for non-specific binding of the secondary antibody, a sample should be incubated with the secondary antibody only.

Are Alexa Fluor® conjugated secondary antibodies applicable to multi-color imaging?

Our Alexa Fluor® conjugated secondary antibodies are ideal for multiplexing. They have been extensively tested to ensure high specificity and sensitivity. Our broad range of Alexa Fluor® conjugates target numerous species allowing for labeling of multiple antigens. See below a table representing our current Alexa Fluor® conjugated secondary antibodies and their characteristics.

Alexa Fluor®Color in vial*Absorption Max (nm)Emission Max (nm)Emission Color**Matching Dyes
405Colourless402421BlueCascade Blue
488Yellow495519GreenCy2, FITC (fluorescein)
555Pink555565OrangeCy3, TRITC (Rhodamine)
568Violet578603Orange/ RedRhodamine Red
594Purple590617RedTexas Red
647Blue650668Far RedAPC, Cy5
680Blue679702Near-IR***Cy5.5, IR680

* 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.

Which Alexa Fluor® conjugated secondary antibodies would you recommend for multi-color imaging?​

When planning a multiplex experiment, it is important to use Alexa Fluor® conjugated secondary antibodies with distinct emission maximum (at least 40 nm difference). The graph below shows the emission spectra of each product we currently offer.

For optimal performance, try using Alexa Fluor® conjugated secondary antibodies without spectral overlap. See below a table showing common combinations for double and triple immunostaining.

ImmunostainingAntibody combination
DoubleAlexa Fluor® 488 and Alexa Fluor® 647
TripleAlexa Fluor® 405 and Alexa Fluor® 555 and Alexa Fluor® 647

How can I minimize cross-reactivity in multiplex experiments?

Cross-reactivity is not expected when using primary antibodies raised in different species (e.g. mouse and rabbit) and secondary antibodies that specifically target each species. If using primary antibodies raised in the same species, they must be of different isotypes (e.g. IgG and IgM). Secondary antibodies that react with specific isotypes will prevent cross-reactivity. Our catalogue includes a broad range of Alexa Fluor® conjugated secondary antibodies targeting diverse species and isotypes.

What does pre-adsorbed mean?

​​Pre-adsorbed secondary antibodies are ideal for eliminating potential antibody species cross-reactivity. Pre-adsorption of the antibody should be performed using serum from the same species as your sample. Thus, species adsorbed antibodies are less likely to interact with endogenous immunoglobulins reducing significantly non-specific binding. We provide a wide range of pre-adsorbed Alexa Fluor® conjugated secondary antibodies.

Feel free to contact our scientific support team at if in need of further assistance.

Please, follow the links below if you would like further information on the Alexa Fluor® conjugated secondary antibodies:

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