Menu

Immunoblot Analysis

Twenty-four hours after conditioning, mice were tested for the expression of place
preference (20min) then sacrificed. Bi-lateral hippocampus was dissected and flash
frozen on dry ice. Tissues were homogenized on ice in 200μl RIPA buffer (4.5mM Tris-
HCl pH7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (Boston
Bioproducts, Ashland, MA) supplemented with Complete mini protease inhibitor cocktail
tablets (Roche Disagnostics, Indianapolis, IN), 1mM EDTA and 1mM PMSF. Samples
were centrifuged at 4°C for 10 min. The supernatant was collected and protein
concentration was measured using DC Protein Assay Kit (Bio-Rad, Hercules, CA). Equal
volumes of laemmli sample buffer (Bio-Rad) were added to each sample aliquot. Prior to
electrophoresis, samples were boiled at 94°C for 5 min and 15μg of total protein was
subjected to electrophoresis on 4-20% SDS–polyacrylamide gels (Bio-Rad). Proteins
were subsequently transferred to Immobilon-FL PVDF Transfer Membrane (Millipore,
Billerica, MA). Membranes were blocked with 3% BSA in 0.1% Tween-supplemented
Tris-buffered saline (TBS-T) for 1 h at room temperature (RT) then probed for NR1
(1:4000; 5704S; Cell Signaling), NR2A (1:4000; 4205S; Cell Signaling), NR2B (1:4000,
06-600; Millipore) and β-tubulin (1:40000; 05-559; Millipore) overnight at 4 °C. After
incubation, membranes were washed 3X for 10 min with TBS-T and then incubated (1hr
at RT) with HRP-linked secondary antibodies. The signal was visualized using an
enhanced chemiluminescent (ECL) substrate for HRP enzyme (Bio-Rad). Band
intensities were quantified using UN-SCAN-IT gel analysis software (v6.1, Silk
Scientific Inc., Orem, UT). Relative density units were expressed as total target
protein/beta-tubulin.

0 Comment

Leave a Reply

Your email address will not be published. Required fields are marked *

x

Hi!
I'm Roberto!

Would you like to get a custom essay? How about receiving a customized one?

Check it out