The frozen CeA tissues were homogenized in ice-cold RIPA lysis buffer containing protease inhibitors and phenylmethylsulfonyl fluoride (Beyotime Biotech, Jiangsu, China). The supernatants were collected after centrifugation at 12,000 g for 15 min at 4°C, and the protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States). Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, China) and transferred onto a nitrocellulose membrane. Then, the membrane was incubated with the following primary antibodies at 4°C overnight: anti-SIRT1 (Cell Signaling Technology, Beverly, MA, United States), and anti-β-actin (Bioworld, Louis Park, MN, United States), followed by incubation with the IRDye 800CW second antibody (Li-Cor, Lincoln, NE, United States). The immunoreactive bands were detected using an Infrared Imaging System (Gene Company Limited, Hong Kong, China) and analyzed with ImageJ software.
Infrared imaging system
Manufactured by Gene Tech
Sourced in Hong Kong
The Infrared Imaging System is a specialized laboratory equipment that uses infrared technology to capture and analyze thermal images. It is designed to detect and measure the heat signatures of various objects or materials within a given environment.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Irdye 800cw second antibody, by LI COR (2 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Anti sirt1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Bioworld Technology (1 mentions)
Sds polyacrylamide gel electrophoresis, by Beyotime (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
A11319, by ABclonal (1 mentions)
A9833, by ABclonal (1 mentions)
A2586, by ABclonal (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
A0208, by ABclonal (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ac004, by ABclonal (1 mentions)
Ab170190, by Abcam (1 mentions)
Ap0060, by Bioworld Technology (1 mentions)
Et1701 54, by Huabio (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Polyvinylidene uoride membrane, by Merck Group (1 mentions)
4 protocols using infrared imaging system
1
Western Blot Analysis of SIRT1 Protein
2
Western Blot Analysis of Spinal Cord
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After pain behavioral tests, the rats were anesthetized and sacrificed by decapitation on day 12 after inoculation of MRMT-1 cells. Lumbar spinal cord was dissected and then homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing a cocktail of protease inhibitors (Sigma). After centrifugation at 12,000 g for 15 min, supernatant was used for Western blot analysis. Protein concentrations were determined by bicinchoninic acid assay method. Equal amounts of protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane and then incubated with the appropriate primary antibodies at 4°C overnight. The following antibodies were used in this study and ABclonal Technology (Wuhan, China): mouse rabbit anti-Drp1 (1:1000, A2586), anti-OPA1 (1:1000, A9833), mouse anti-glial fibrillary acidic protein (GFAP) (1:1000, A0014), rabbit anti-caspase 3 (1:1000, A11319), rabbit anti-Bcl-2 (1:1000, A0208), rabbit anti-β-actin (1:5000, AC004). Horse radish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Abcam) were used to visualize the primary antibodies. Infrared Imaging System (Gene Company Limited, Hongkong, China) was applied to detect immunoreactive bands.
3
Western Blotting Analysis of Spinal Cord Proteins
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Western blotting analysis was performed as we described previously [26 (link)]. Briefly, the frozen spinal cords were homogenized in lysis buffer (Beyotime Biotech Inc., Jiangsu, China) containing a mixture of protease inhibitors and phenylmethylsulfonyl fluoride. The supernatant was collected after centrifugation at 12000 g under 4°C for 15 min and the protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Massachusetts, USA). Next, the protein samples were separated in equal quantities by SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Merck Millipore, Massachusetts, USA). After blocking with 5% skim milk powder, the membrane was incubated with appropriate primary antibodies that against SIRT3 (1 : 1000, D22A3, Cell Signaling Technology, USA), CypD (1 : 1000, ab170190, Abcam, UK), Ac-CypD-K166 (1 : 1000, WG-00545P, ABclonal, China), MnSOD (1 : 1000, ET1701-54, HUABIO, China), or β-actin (1 : 1000, AP0060, Bioworld, Louis Park, USA) overnight at 4°C, followed by incubation with the IRDye 800CW second antibody (Li-Cor, Lincoln, Nebraska, USA). Immunoreactive bands were detected using an Infrared Imaging System (Gene Company Limited, Hong Kong, China).
4
Protein Expression and Quantification
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Protein concentration of the sample was determined using a BCA protein assay kit (Thermo Scienti c, Rockford, IL, USA). The protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene uoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 2% milk powder solution for 60 min. Primary antibodies including anti-NLRP1 (Abcam Company, USA, 1:1000), anti-cleaved-caspase-1 (Bioworld Company, USA, 1:1000), anti-PAR-1 (Sigma-Aldrich Company, USA, 1:1000), anti-RAGE (Abcam Company, UK, 1:1000), anti-NF-κB p65 antibody (A nity Biosciences, UAS, 1:1000), and anti-NF-κB p-p65 (A nity Biosciences, UAS, 1:1000) were incubated overnight at 4°C, followed by IRDye puri ed goat anti rabbit IgG(H+L) secondary antibodies (Li-Cor Inc., Lincoln, NE), respectively. Immunoreactive blots were detected using Infrared Imaging System (Gene Company Limited, Hong Kong, China), and the signal densities on the blots were measured with Image J software; meanwhile, they were normalized using rabbit anti-β-actin antibody (Bioworld Technology, St. Louis, USA) or rabbit anti-GAPDH antibody (ABclonal Biotechnology Co., Ltd., USA) as an internal control.
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