Analysis Note

ControlRat brain tissue human brain lysate

Application

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

Research CategoryNeuroscience

Research Sub CategoryNeurotransmitters & Receptors

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.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

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.

Immunogen

Purified rat brain glutamic acid decarboxylase.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

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

Physical form

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).

Format: Purified

Specificity

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).

Storage and Stability

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.

Target description

65 kDa