This article details a protocol for isolating and analyzing glutamic acid decarboxylase (GAD) isoforms from human brain tissue. The method involves homogenization, centrifugation, and electrophoresis to visualize protein bands.
Begin with a human brain tissue in a lysis buffer.
Homogenize the tissue with a homogenizer to release proteins, including glutamic acid decarboxylase, or GAD enzyme isoforms, essential for brain function.
Incubate the homogenate on ice, then centrifuge to pellet the debris. Collect the protein-containing supernatant.
Add a loading buffer containing a detergent and a reducing agent to denature the proteins.
Load this sample and a molecular marker onto a gradient SDS-PAGE gel and electrophorese to separate the proteins into distinct bands.
Electroblot the proteins from the gel onto a membrane sheet.
Add a blocking agent to prevent nonspecific binding.
Incubate with GAD-isoform-specific primary antibodies that bind to their receptive GAD isoforms.
Wash to remove unbound antibodies.
Incubate with fluorescently labeled secondary antibodies.
Wash again and visualize the protein bands on an imaging system.
Detect the GAD bands through fluorescence and confirm their molecular weight differences using the marker.
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