Alexa Fluor 555 Secondary Fluorescent Antibody for Immunofluorescence

January 12, 2016

Food Science and Human Nutrition
Colorado State University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

Alexa Fluor 555

Catalog Number:

ab150114

This secondary was used in conjunction with mouse primary antibodies to detect alpha tubulin and acetylated alpha tubulin under a fluorescent microscope using the Cy3 filter cube

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

H4IIE rat hepatoma cells

Primary Incubation

1:50 primary antibody (mouse alpha-tubulin), 1% BSA, in PBST (PBS+ 0.1% Tween 20)

Blocking Agent

1% BSA, 22.52 mg/ml glycine in PBST (PBS+ 0.1% Tween 20)

Secondary Incubation

1:200 Goat anti-mouse Alexa Fluor 555, 1% BSA, in PBST (PBS+ 0.1% Tween 20)

Tertiary Incubation

NA

Detection

Olympus BX63 Fluorescent microscope

Results Summary

This secondary antibody was one of the brightest I have worked with under the microscope. The exposure time of my photos were in the 60-100 millisecond range which eliminates a lot of photobleaching which I have had issues with in the past using longer exposures. Very little background with this antibody and it is very efficacious as it tagged 90-100% of the cells in my sample.

Additional Notes

None

Image Gallery

Summary

The Good

Little background signal. Very efficacious.

The Bad

No protocol. Abcam has a standard protocol that they suggest and that worked, but I had to dig around to find it. Also Abcam shows this secondary fluorescing as yellow. Before purchasing, know the wavelength specifications of the filter cubes you have.

The Bottom Line

Great secondary antibody for immunofluorescence. Easily detectable bright red under Cy3 filter cube.

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