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High sensitivity ecl

Manufactured by Bio-Rad
Sourced in United States

High-sensitivity ECL is a chemiluminescent detection system for Western blotting applications. It provides a high-intensity signal for the detection of low-abundance proteins.

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2 protocols using high sensitivity ecl

1

Evaluating mGluR2 Expression in Nucleus Accumbens

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To investigate whether mGluR2 protein expression was downregulated in the nucleus accumbens shell after two weeks of CIE, a separate cohort of mice underwent ethanol self-administration and CIE exposure following the protocol described above. Mice were sacrificed after two weeks of CIE via rapid decapitation under isoflurane anesthesia. Nucleus accumbens shell was microdissected from tissue, sonicated in LDS, and stored at −20 °C until Western blot analysis. After transfer, total protein was assessed using Swift Membrane Stain (G-Biosciences). Membranes were probed using a selective mouse anti-mGluR2 antibody (Abcam; 1:1000) and visualized using a HRP-conjugated goat anti-mouse secondary antibody (Southern Biotech; 1:5000) with BioRad high-sensitivity ECL. mGluR2 protein was normalized to total protein to control for loading.
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2

Western Blot and qPCR Analysis Protocol

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RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was used for cell and tissue lysis. Protein was extracted by centrifugation at 12,000 g and detected by BCA assay at 562 nm. A rapid gel preparation kit (Beyotime Biotechnology, Shanghai, China) was used to configure the gel distribution at 130 V constant voltages and 240 mA constant current elec-troconversion. Primary antibody was incubated at 4 °C overnight. Secondary antibody was incubated at 37 °C for 1 hour. Phosphate buffered saline was used to wash test strips three times for five minutes each. High-sensitivity ECL (Bio-Rad, USA) solution was used for the exposure strip. ImageJ software was used to analyze results. RNA extraction and qPCR were performed using RNAiso reagent and a PrimeScript™ Reverse Transcriptase kit (Takara, Shiga, Japan) following manufacturer instructions (Par. 1-7).
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