CDKL5 Western Blot Antibody

September 13, 2017

Molecular Physiology and Biophysics
Baylor College of Medicine
Other

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Company:

Sigma/ Atlas Antibodies

Product Name:

Anti-CDKL5 antibody produced in rabbit

Catalog Number:

hpa002847

Our lab is interested in developing mouse models of CDKL5 disorder. We needed an antibody that would allow us to determine the CDKL5 expression in mice possessing targeted mutations (knock out and knock in) in the Cdkl5 locus. We tested this antibody for both Immunofluorescence and Western Blot detection of Cdkl5. It is not a very specific antibody and appears to bind promiscuously to several other proteins with apparently similar epitopes. However, it does bind to a Western Blot band that appears to be wildtype Cdkl5.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Mouse brain (frontal cortex)

Primary Incubation

1:500 4C overnight in 5%BSA, 0.1% Tween-20, 1xPBS

Blocking Agent

5%BSA in 1x PBS

Secondary Incubation

1:10k Licor 800 conjucated anti-rabbit prepared in 5%BSA, 1xPBS

Tertiary Incubation

N/A

Detection

Licor imager

Results Summary

Several nonspecific bands are visible, but a band near the expected molecular weight of Cdkl5 is visible in wildtype samples and absent in mutant samples. (Note - the binding to several non-specific bands would explain the difficulty in getting immunofluorescence staining that is specific to cdkl5 status. In our hands, there was too much nonspecific signal that prevented our ability to distinguish wiltype from mutant samples)

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

It does detect a band that is probably Cdkl5

The Bad

It also detects several other non-specific proteins

The Bottom Line

So far, there is a limited selection of Cdkl5 antibodies that work well. Several have problems with detecting non-specific signals. While this antibody has a similar problem, it is just good enough to get a useful western blot result regarding Cdkl5 expression.

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