An Essential Piece of the MeCP2 Antibody Tool Kit

May 19, 2016

Molecular Physiology and Biophysics
Baylor College of Medicine
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Company:

Sigma-Aldrich

Product Name:

Monoclonal Anti-MeCP2 antibody produced in mouse

Catalog Number:

M7443-200UL

Browse Similar Products: MeCP2 Immunofluorescence Antibodies

Our lab is interested in how MeCP2 (the gene/protein linked to Rett Syndrome) functions within the brain and how loss of function creates pathology. We needed an antibody to help us detect MeCP2 truncation mutations. This antibody will stain MeCP2 in the cells of mouse and rat tissue (probably human too, but I haven't tried that yet). The signal is usually good and specific to the nucleus (where MeCP2 should be), with the main drawbacks being the standard problems with mouse antibodies: the secondary antibody will also tend to mark the blood vessels. Because of the peptide used to generate this antibody, our lab uses it to detect the N-terminal region of Mecp2. It is one of the few antibodies that we've come across with good staining signal and specificity for the N-terminal region of MeCP2.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Fresh frozen mouse brain (30 minute PFA fixation on slide, 15 minute permeabilization with 1xPBS 0.3% TritonX100)

Primary Incubation

Mouse anti MeCP2 antibody diluted 1:500 in 1xPBS 10% donkey serum, incubated 16 hours at room temperature

Blocking Agent

1xPBS with 10% Donkey Serum

Secondary Incubation

Donkey anti mouse-Alexa 488 conjugated antibody from JaxIR diluted 1:500 in 1xPBS 10% donkey serum, incubated 4 hours at room temperature

Tertiary Incubation

NA

Detection

Immunofluorescent microscopy with apotome (Alexa 488 channel)

Results Summary

The antibody is able to recognize the N-terminal region of Mecp2. It stains the nuclei of wild type neurons, as well as cells that are expressing premature truncation mutations of Mecp2. In animals that completely lack Mecp2 expression, there is no nuclear staining signal. Unfortunately, in mouse tissue it is common to have to deal with the secondary antibody also detecting other sources of mouse antibodies (those that may have been left behind in the blood vessels). This 'non-specific' signal can usually be differentiated by Mecp2-specific staining by morphology and intensity of signal.

Additional Notes

None

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Summary

The Good

It detects the N-terminal region of Mecp2 in mouse and rat

The Bad

Secondary antibody to detect this may also stain blood vessels

The Bottom Line

This antibody adds flexibility when performing immunofluorescent histology for multiple markers where you also need to stain for MeCP2 (the rabbit [07-013] and chicken [abe171] anti mecp2 offered by millipore complete the set that our lab uses). The epitope specificity for the n-terminal region can be useful when an experiment requires distinguishing between the n and c terminal regions of MeCP2

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