Decent Secondary Antibody From Thermo Fisher for IF Detection of Proteins

February 22, 2024

IKP/RBCT
Robert Bosch Gesellschaft für medizinische Forschung mbH
Graduate Student

Overall

Quality of Results

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Company:

Thermo Fisher Scientific

Product Name:

Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 405

Catalog Number:

A48254

Clone Number:

N/A

This Alexa Fluor Plus 405 secondary antibody is a well-working option to stain your proteins using immunofluorescence and detection in the blue wavelength segment (em. 404 nm / ex. 455 nm).The antibody is productive and the staining is stable. Quality depends largely on the primary rabbit antibody used.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

PFA-fixed and permeabilised (human) cells e.g. Hela

Primary Incubation

1:100 for 2 h in Blocking

Blocking Agent

1 % BSA + 2 % FCS in PBS

Secondary Incubation

1:500 for 1h in Blocking

Tertiary Incubation

N/A

Detection

Confocal laser scanning microscopy (405 nm laser)

Results Summary

Using the 405 nm laser or respective filter cube on a fluorescence microscope, the signal is clearly detectable around 455 nm. Fluorescence can be easily combined with green, red or farred dyes without high crosstalk. Brightness and signal quality depends on the primary rabbit antibody used.

DOI or PMID #

N/A

Additional Notes

Antibody can be kept at 4°C for several weeks/months without major decrease in quality, spin down before usage to avoid conglomerates, initially a concentration of 1:100 can be tested with new primary antibodies.

Image Gallery

Summary

The Good

Stable and bright signal.

The Bad

Pricey.

The Bottom Line

The Alexa Fluor Plus 405 secondary antibody against rabbit primaries is a decent option for your daily IF staining. Although signal quality depends on your primary antibody, it generally is reliable and easy to combine with additional dyes.

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