Samples of brain lysates were prepared as described previously32 (link). Briefly, brain tissue was incubated in RIPA lysis buffer (150mM NaCl, 50mM Tris pH 8, 5mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) with protease and phosphatase inhibitors (1:100, Calbiochem #539131 and #524625, respectively). Samples were triturated, rotated at 4°C for 30 minutes then spun down at 14,000 rpm for 10 minutes at 4°C. The supernatant was collected and cell debris discarded. Protein concentration was measured using a BCA kit. To measure FAAH levels, 10mg of forebrain lysate was resolved by SDS/PAGE electrophoresis (NuPAGE 10% Bis-Tris gel; Invitrogen #NP0315) and probed with antibodies: mouse monoclonal anti-FAAH (Abcam, ab54615; 1:500) and goat polyclonal anti-actin (Santa Cruz, sc-1616HRP; 1:5000) were used. As a positive control, 293T cells purchased from American Type Culture Collection (ATCC) were transfected with 1mg mouse FAAH cDNA clone (Origene, NM_010173), and lysates were processed in parallel. Forebrain lysate from FAAH knockout mice5 (link) were used as a negative control.
Mouse faah cdna clone
Manufactured by OriGene
The Mouse FAAH cDNA clone is a laboratory product that contains the complementary DNA (cDNA) sequence encoding the mouse Fatty Acid Amide Hydrolase (FAAH) enzyme. FAAH is an integral membrane serine hydrolase responsible for the degradation of endocannabinoids and other fatty acid amides. The clone can be used for various research applications, such as the study of the FAAH enzyme and its role in biological processes.
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2 protocols using mouse faah cdna clone
1
Brain Lysate Preparation and FAAH Analysis
2
Brain Lysate Preparation and FAAH Analysis
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Samples of brain lysates were prepared as described previously32 (link). Briefly, brain tissue was incubated in RIPA lysis buffer (150mM NaCl, 50mM Tris pH 8, 5mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) with protease and phosphatase inhibitors (1:100, Calbiochem #539131 and #524625, respectively). Samples were triturated, rotated at 4°C for 30 minutes then spun down at 14,000 rpm for 10 minutes at 4°C. The supernatant was collected and cell debris discarded. Protein concentration was measured using a BCA kit. To measure FAAH levels, 10mg of forebrain lysate was resolved by SDS/PAGE electrophoresis (NuPAGE 10% Bis-Tris gel; Invitrogen #NP0315) and probed with antibodies: mouse monoclonal anti-FAAH (Abcam, ab54615; 1:500) and goat polyclonal anti-actin (Santa Cruz, sc-1616HRP; 1:5000) were used. As a positive control, 293T cells purchased from American Type Culture Collection (ATCC) were transfected with 1mg mouse FAAH cDNA clone (Origene, NM_010173), and lysates were processed in parallel. Forebrain lysate from FAAH knockout mice5 (link) were used as a negative control.
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