Assays

PKA Assay

1. Homogenate a pair of hippocampi in 200 ul [5 mM EDTA, 50 mM Tris 7.5, protease inhibitor cocktail, 5 mM theophylline].

2. Mix 10 ul homogenates, 20 ul [50 mM Tris 7.5, 7.5 mM theophylline], then add 10 ul 32p/substrate, 4 ul ATP to 1 ml [200 uM kemptide, 400 uM ATP, 40 mM MgCl2, 1 mg/ml BSA, 50 mM Tris 7.5]

3. 30 degrees for 5 min

4. Spot 20 ul on phosphocellulose disc

5. Immerse in 1% (v/v) H3PO4 (at least 10 ml/sample), rock at 127 for 3-5 min, repeat one more acid wash

6. Wash with 2X H2O 3-5 min/each

7. Count p32, also count 10 ul of the original reaction and do protein assay

Stock of kemptide = 2 mM

Phosphocellulose disc: whatman P81 CAT No: 3698325

32p-r-ATP: Perkin Elmer cat #: BLU002H

When doing Ca+ stimulated pkA assay: use 2 ug calmodulin/reaction, stock 1 mg/ml

cAMP Accumulation Assay

1. The evening before actual assay, load cells with [3H]-adenine. for cell line cells, 1-2 uCi/ml is enough. Higher for neurons (~3 uCi/ml). Incubate overnight.

2. Aspirate media containing 3H-adenine. Rinse with PBS or media (optional). Pre-incubate with treatment solution in the presence of IBMX for at least 30'-60' (DMEM or buffered solution okay). Then treat for 5'-15'.

3. At the end of treatment, stop by aspirating treatment mix and add 1 ml 5% TCA per well (can be kept in 4 degrees C for days).

4. Run columns.

Keywords.

Adenylyl cyclase (AC), amyloid precursor protein (APP), Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), cAMP, cAMP-responsive element (CRE), CREB, extra-cellular regulated protein kinase (ERK), FE65, long-term potentiation (LTP), long-term depression (LTD), memory formation, neuroplasticity, local protein synthesis.