Anti-Glutamate Decarboxylase Antibody, 65 kDa isoform, clone GAD-6

Gutamic acid decarboxylase (GAD; E.C. 4.1.1.15) is the enzyme responsible for the conversion of glutamic acid to gamma-aminobutyric acid (GABA), the major inhibitory transmitter in higher brain regions, and putative paracrine hormone in pancreatic islets. Two molecular forms of GAD (65 kDa and 67 kDa, 64% aa identity between forms) are highly conserved and both forms are expressed in the CNS, pancreatic islet cells, testis, oviduct and ovary. The isoforms are regionally distributed cytoplasmically in the brains of rats and mice (Sheikh, S. et al. 1999). GAD65 is an ampiphilic, membrane-anchored protein (585 a.a.), encoded on human chromosome 10, and is responsible for vesicular GABA production. GAD67 is cytoplasmic (594 a.a.), encoded on chromosome 2, and seems to be responsible for significant cytoplasmic GABA production. GAD expression changes during neural development in rat spinal cord. GAD65 is expressed transiently in commissural axons around E13 but is down regulated the next day while GAD67 expression increases mostly in the somata of those neurons (Phelps, P. et al. 1999). In mature rat pancreas, GAD65 and GAD67 appear to be differentially localized, GAD65 primarily in insulin-containing beta cells and GAD67 in glucagon-containing (A) cells (Li, L. et al. 1995). GAD67 expression seems to be particularly plastic and can change in response to experimental manipulation (for example neuronal stimulation or transection) or disease progression and emergent disorders like schizophrenia (Volk et al., 2000). Colocalization of the two GAD isoforms also shows changes in GAD65/GAD67 distributions correlated with certain disease states such as IDDM and SMS.

Recognizes the lower molecular weight isoform of the two GAD isoforms identified in brain (Gottlieb, et al., 1986; Chang & Gottlieb, 1988). This monclonal antibody can be used for immunohistochemical localization in brain or pancreas. Anti-GAD has also been used to label purified GAD on Western blots (Chang & Gottlieb, 1988).

Purified rat brain glutamic acid decarboxylase.

Anti-GAD Antibody. 65 kDa isoform, clone GAD-6 detects level of Glutamate Decarboxylase & has been published & validated for use in IH & WB.

Immunohistochemistry: ( 1 μg/mL * See protocol)

Western blot

Optimal working dilutions must be determined by end user.

APPLICATION NOTES FOR MAB351

IMMUNOHISTOCHEMISTRY

1) Perfuse rats with 100 mM phosphate buffer, pH 7.4 containing 1% paraformaldehyde, 0.34% L-lysine and 0.05% m-periodate (1% PLP).

2) Postfix brains in 1% PLP for 1-2 hours.

3) Transfer brains to 100 mM phosphate buffer containing 30% sucrose and gently agitate on a shaker platform at +4°C for 48-60 hours.

4) Using a sliding microtome, cut 30 mm sections of frozen cerebellum. As the sections are cut, collect them in a vial of cold 100 mM phosphate buffer.

5) Incubate sections in PBS containing 1.5% normal serum and 0.2% Triton X-100 for 30 minutes.

6) On a shaker platform, incubate sections with MAB351 (diluted 1 μg/mL in PBS containing 1.5% normal serum and 0.2% Triton X-100) for 12-36 hours at +4°C.

7) On a shaker platform, rinse sections eight times, 10-15 minutes per rinse, in PBS.

8) Detect with standard secondary antibody detection system (PAP, ABC, etc.).

9) Mount sections, dehydrate, and apply coverslips.

Research Category
Neuroscience

Research Sub Category
Neurotransmitters & Receptors

65 kDa

Format: Purified

Protein A purified

Purified immunoglobulin. Lyophilized from 10 mM potassium phosphate, 70 mM sodium chloride, pH 7.4, 0.02% sodium azide. Reconstitute with 100 μL of sterile water (1 mg/mL).

Maintain lyophilized material at -20°C for up to 12 months. After reconstitution maintain frozen at -20°C in undiluted aliquots for up to 6 months. Avoid repeated freeze/thaw cycles.

Control
Rat brain tissue

human brain lysate

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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