Panorama® Neurobiology Array

By: Carolyn L. Crankshaw, BioFiles v7 n2, 2011, 21–22

BioFiles Volume 7, Number 2 — Neurodegenerative Diseases

Download BioFiles v7 n2 (13.0 Mb PDF)

Antibody microarrays are used for profiling complex protein samples and for assessing differential protein expression. The Panorama Ab Microarray kits, consisting of a series of arrays that cover various biological pathways such as Cell Signaling, Gene Regulation and MAPK/PKC pathways, have already proven to be useful tools for such applications. A new antibody array consisting of 224 neurobiology related antibodies has been developed. Refer to Figure 1 for antibody distribution. Using this array, we compared brain tissue of newborn and adult rats in order to study protein expression during rat brain development. We identified several proteins that changed with age. The outline of the experimental procedure is highlighted in Figure 2.

Distribution of neurospecific antibodies comprising the Panorama Neuorobiology Array.

Figure 1 – Distribution of neurospecific antibodies comprising the Panorama Neuorobiology Array.

Neurobiology Antibody Array Procedure

Figure 2 – Neurobiology Antibody Array Procedure.

Expression of proteins were prepared from rat newborn (4 days) and adult (2 month) brains using Extraction/Labeling Buffer (as supplied in Panorama® Neurobiology kit), and labeled with Cy3. Equal amounts of labeled extracts (20 µg/ml) were incubated on the Neurobiology slides. Proteins on left (indicated in green), demonstrate decline in development. Proteins on right (indicated in red, i.e. Synaptopodin, Ubiquitin C-terminal Hydolase L1, and alpha/beta SNAP), are associated with increase with age. Figure 3.

Differential Expression between Newborn and Adult Rats

Figure 3 – Differential Expression between Newborn and Adult Rats.

Array results were confirmed by immunoblotting. Equal amounts of protein extract (20 µg per lane) from rat adult (A) or newborn (N) brains were separated by SDS-PAGE and blotted onto nitrocellulose membrane. The proteins were probed with the monoclonal or polyclonal antibodies corresponding to the array and visualized using chemiluminescence.

Further studies using the Panorama Neurobiology array have been performed identifying several additional proteins that changed with age. For example, PINK1 was shown to be elevated in adult versus newborn brains. Further evaluation of the new Panorama Antibody Neurobiology Array was performed using brain tissue from the knockout rat model PARK2 -/-. The array has demonstrated that indeed this gene expression is being ablated in the knockout rats versus wild type siblings, a result validated by immunoblotting (data not shown).

In conclusion, the new Panorama Neurobiology array has shown to be a promising and useful tool for highthroughput screening (HTS) of protein level changes in neuronal development and neurodegenerative disease.

 

Materials

     
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