Lung tissues were homogenized in RIPA in the presence of a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail (Roche). The protein concentrations were determined by a BCA kit (Pierce, Rockford, IL, USA). Equal concentrations of protein (25 μg) were separated by SDS-PAGE on 12% acrylamide gels. The primary antibodies used were as follow: NLRP3 (Adipogen, San Diego, CA, USA), caspase 1 (p20) (Adipogen), IL-1β, pro-IL-1β (R&D Systems, Minneapolis, MN, USA), TLR2 (Millipore, Billerica, MA, USA), AANAT (Sigma), ASMT (Abcam, Boston, MA, USA) and GAPDH (KANGCHEN Biotech, Shanghai, China) for 1 h at 37°C, followed by at 4°C overnight. Blots were washed thrice with TBST and probed with appropriate secondary antibodies for 1 h at room temperature. After washing three times, the immune-reaction was analyzed using the ECL detection system (Pierce). The band density was determined by ImageJ software (NIH, Bethesda, MD, USA).
Aanat
Manufactured by Merck Group
Sourced in United States
AANAT is a laboratory equipment product manufactured by Merck Group. It is an enzyme that plays a key role in the biosynthesis of melatonin, a hormone important for regulating the sleep-wake cycle. The core function of AANAT is to catalyze the conversion of N-acetylserotonin to melatonin.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Kangchen Biotech (2 mentions)
Caspase 1 p20, by Adipogen (1 mentions)
Pro il 1β, by R&D Systems (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Nlrp3, by Adipogen (1 mentions)
Bmal1, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Promega (1 mentions)
2 protocols using aanat
1
Western Blot Analysis of NLRP3 Inflammasome
2
Western Blot Analysis of Circadian Proteins
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Lung tissue was homogenized on ice using Tissuelyser (Tiss-24, Shanghai Jingxin Industrial Development Co., Ltd, Shanghai, China)) with RIPA buffer lysate to extract the total protein. Equal amounts of protein were separated by a 12% SDS-PAGE and then transferred to PVDF membrane. The membranes were blocked in 5% non-fat milk for 1 h. The primary antibodies: Bmal1, Per1, Clock, Timeless, Cry1 and Cry2 (all from abcam, Boston, MA, USA), ASMT (abcam), AANAT (Sigma), Mel-1A/B–R (Santa Cruz Biotechnology, CA, USA), GAPDH (KANGCHEN Biotech, China) were incubated respectively at room temperature for 1 h and 4 °C overnight. The next day, after being washed with TBST thrice and incubated with appropriate HRP-conjugated secondary antibodies (Promega, madison, wi, USA) for 1 h, bands were washed with TBST for thrice again. Then, enhanced chemiluminescence (ECL, Biosharp, Beijing, China) and Chemiluminescence imaging system (Qing Xiang, Shanghai, China) were used to detect the protein immunostaining. The density of protein bands was obtained by using Image J 1.48v software (NIH, Bethesda, MD, USA).
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