After cell collection and total protein extraction, the proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto poly vinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, washed three times with tris-buffered saline Tween (TBS-T) solution and incubated with a primary antibody at 4 °C overnight. Subsequently, the membranes were incubated with anti-NLRP3 (1:1000 Abcam, Cambridge, MA, USA), ASC (1:1000 Affinity, America), caspase-1 (1:1000 Affinity, America), P-caspase-1 (1:1000 Abcam, MA, USA), iNOS and Arg-1 (1:1000 Zen BioScience, China) primary antibodies. The membranes were washed thrice with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (Sparkjade, China) for 2 h at room temperature. After subsequent washing with TBS-T solution thrice, protein bands were detected using an ECL (Affinity, America) kit. Finally, the band intensities were quantitatively analysed using ImageJ analysis software.
Caspase 1
Manufactured by Affinity Biosciences
Sourced in United States
Caspase-1 is a laboratory enzyme used in research. It plays a key role in the inflammatory response and programmed cell death processes. This product provides a reliable tool for studying these fundamental cellular mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Affinity Biosciences (4 mentions)
Nlrp3, by Affinity Biosciences (4 mentions)
Nlrp3, by Abcam (3 mentions)
Enhanced chemi luminescence (ecl), by Affinity Biosciences (2 mentions)
P iκbα, by Affinity Biosciences (2 mentions)
β actin, by Affinity Biosciences (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
4 hne, by Alpha Diagnostic (2 mentions)
Gsdmd, by Affinity Biosciences (2 mentions)
Anti nlrp3, by Abcam (1 mentions)
P enos, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 1, by Cell Signaling Technology (1 mentions)
Detergent compatible bradford protein assay kit, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Caspase 3, by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Cleaved caspase 1, by Affinity Biosciences (1 mentions)
α tubulin, by Affinity Biosciences (1 mentions)
S8639, by Selleck Chemicals (1 mentions)
14 protocols using caspase 1
1
Western Blot Analysis of NLRP3 Inflammasome
2
Evaluating Inflammasome Activation in Mouse Aortic Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse aortic endothelial cells pre-treated with different treatments were investigated. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL, United States) and total protein concentration was adjusted to detect the expressions of NALP3 (Cell Signaling Technology, Beverly, MA, United States), ASC (Abcam, Cambridge, United Kingdom), caspase-1 (Affinity, Cincinnati, OH, United States), cleaved caspase-1 (Cell Signaling Technology, Beverly, MA, United States), p-eNOS and e-NOS (Abcam, Cambridge, United Kingdom) by western blot. Results were analyzed using Quantity One software. The concentration of IL-1β in cell culture supernatants was determined using an ELISA kit (R&D Systems, Minneapolis, MN, United States) in accordance with the manufacturer’s instructions.
3
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lung tissues were ground under liquid nitrogen, transferred to RIPA buffer (Beyotime Biotechnology Co. Ltd., Shanghai, China) for lysis, and centrifuged to collect the supernatant. The total protein concentration was determined using the Detergent Compatible Bradford Protein Assay Kit (Beyotime Biotechnology Co. Ltd., Shanghai, China). The protein concentration of the samples was adjusted for consistency. A loading buffer was then added and boiling was done to denature the protein. Samples containing 20 μg protein were separated using 10% or 12% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with tris-buffered saline (TBS) containing 5% non-fat milk for 1 h. The PVDF membranes were then incubated with primary antibodies against NLRP3, ASC, Caspase-1, cleaved-Caspase-1, BAX, Bcl-2, Caspase-3, or α-tubulin (Affinity Biosciences LTD, USA) overnight at 4°C. After washing three times with a TBS Buffer containing 0.1% Twain 20, the membranes were incubated with a secondary antibody (HRP-conjugated) for 1 h at room temperature. The signals of ECL chemiluminescence intensity were captured using the FluorChem FC3 system (Protein Simple, USA). The optical density of the protein bands was analyzed using ImageJ software (NIH, Bethesda, USA).
4
Investigating Inflammasome Signaling Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RhS100A9, CLI-095, rhIL-1β and DPI were purchased from Abcam (ab95909), Invivogen (CA92121), Pepro Tech (200-01B) and Selleck (S8639), respectively. Primary antibodies were obtained from the following manufacturers: p53, p21, and p16 from Cell Signalling Biotechnology (Danvers, MA, USA); NLRP3 from Abcam (Cambridge, MA, USA); and GAPDH, Caspase-1, and IL-1β from Affinity Biosciences. Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) and FITC conjugated anti-rabbit IgG (H + L) secondary antibodies were purchased from Thermo Scientific (Rockford, IL, USA) and Affinity Biosciences, respectively. HRP conjugated secondary antibodies were purchased from Affinity Biosciences.
5
Anti-inflammatory Mechanism of Aspirin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aspirin Tablets were purchased from Bayer S.p.A (Viale Certosa, Milano, Italy). Adenosine diphosphate (ADP) was purchased from TargetMol (Wellesley Hills, MA, USA). BoxA was purchased from HMGBiotech (Via Olgettina, Milano, Italy). Carrageenan was purchased from Sigma-Aldrich (St Louis, MO, USA). ECL was purchased from Affinity Biosciences (OH, USA). GQP was purchased from Huahong Pharmaceutical Co. Ltd (BatchNo. 20191002; Liuzhou, China). CFSE and PGE1 were purchased from Cayman Chemical (Ann Arbor, MI, USA). N4-Acetylcytidine triphosphate sodium was purchased from Good Laboratory Practice Bioscience (Montclair, California, USA). Thrombin was provided from Macklin Biochemical (Shanghai, China).
Rabbit anti-HMGB1 (Cat# 6893S), IκBα (Cat# 4060L), NF-κBp65 (Cat# 8242T), p-NF-κBp65 (Cat# 3033T), NLRP3 (Cat# 15101S) and HRP-linked Antibody (Cat# 7074S) were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin (Cat# AF7018), Caspase 1 (Cat# AF5418) and p-IκBα (Cat# AF2002) were purchased from Affinity Biosciences.
Rabbit anti-HMGB1 (Cat# 6893S), IκBα (Cat# 4060L), NF-κBp65 (Cat# 8242T), p-NF-κBp65 (Cat# 3033T), NLRP3 (Cat# 15101S) and HRP-linked Antibody (Cat# 7074S) were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin (Cat# AF7018), Caspase 1 (Cat# AF5418) and p-IκBα (Cat# AF2002) were purchased from Affinity Biosciences.
6
TLR4-Mediated Inflammatory Pathway Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer containing protease inhibitors was used to extract proteins from rat spinal cord tissues and BV-2 cells. Protein concentrations were then determined using a BCA Protein Assay Kit (Abcam). Protein samples (20 μg/lane) were separated via 10% SDS-PAGE and the resolved proteins were transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin at room temperature. After blocking, membranes were incubated overnight at 4 °C with primary antibodies against TLR4 (1:1000; Abcam), MyD88 (1:1000; Abcam), p65 (NF-κB) (1:1000; Abcam), p-p65 (phospho-NF-κB) (1:1000; Cell Signaling), NLRP3 (1:1000; Abcam), ASC (1:1000; Affinity Biosciences), caspase 1 (1:1000; Affinity Biosciences), N-GSDMD (1:1000; Abcam), and GAPDH (1:1000; Abcam). Thereafter, they were washed three times with Tris-buffered saline Tween-20. Subsequently, an HRP-conjugated IgG secondary antibody (1:5000; Santa Cruz, Waltham, MA, USA) was added and membranes were incubated at room temperature for 1 h. GAPDH was used as the internal reference. An enhanced chemiluminescence detection kit (Thermo Fisher Scientific) was used to detect the bands, which were then quantified using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Silver Spring, MD, USA).
7
Metformin Modulates Gut Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Metformin (MET) was obtained from Sino-American Shanghai Squibb Pharmaceuticals, Ltd. (Shanghai, China). All assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse insulin (CSB-E05071m), LPS (CSB-E13066m), IL-18 (CSB-EO4609) and IL-1β (CSB-E08054) ELISA kits were provided by HuaMei Co. (Shanghai, China). Tissue lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO) containing a protease/phosphatase inhibitor cocktail. The following primary antibodies were used: TLR4 (Affinity, San Francisco, CA, AF7017), NF-κB p65 (CST, Boston, MA, #8242), phospho-NF-κB p65 (CST, Boston, MA, #3033), NLRP3 (Abcam, Cambridge, UK, ab263899), ASC (Affinity, San Francisco, CA, AF6304), caspase-1 (Affinity, San Francisco, CA, AF5418), ZO-1 (CST, Boston, MA, #8193), occludin (Abcam, Cambridge, UK, ab216327) and β-actin (CWBIO, Beijing, China, CW0096). The secondary antibodies used were goat anti-rabbit IgG (CWBIO, Beijing, China) and goat anti-mouse IgG (CWBIO, Beijing, China).
8
Liver Tissue NLRP3 Inflammasome Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis of liver tissues was performed according to standard procedures using specific antibodies including NLRP3, caspase-1, IL-1β and IL-18 (Affinity Biosciences, USA). β-actin was used as an internal control. After reacting with the secondary antibody, proteins were detected with an enhanced chemiluminescence (ECL) Western blotting detection reagent (Millipore, USA) and visualised on a FluorChem E ultra-sensitive automatic imaging analysis system (ProteinSimple, USA).
9
Cardiomyocyte Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heart tissues and AC16 cardiomyocytes were homogenized in ice-cold 1× RIPA lysis buffer, supplemented with a protein inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) to obtain total protein. Total protein was separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with the following antibodies: 3-NT (Millipore, Billerica, MA, USA), 4-HNE (Alpha Diagnostic International, SAN Antonio, TX, USA), NLRP3, caspase1, IL-1β, TXNIP (Affinity, Jiangsu, China) and β-actin (Santa Cruz, Dallas, TX, USA). After washing unbound antibodies, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz, Dallas, TX, USA) for 1 h at room temperature. Specific bands were visualized using an enhanced chemiluminescence detection kit (ECL) and Gel Documentation 2000 system (Bio-Rad, Hercules, CA, USA). Densitometric analysis of protein bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
10
Western Blotting of Neuroinflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein expression levels of p-p38, p38, p-IκBα, Caspase1, COX2, and iNOS were detected by western blotting. The total protein concentration of the brain tissues was analyzed with a BCA kit (Sigma, CA, USA). The blots were separately probed with rabbit antibodies against p-p38 (1 : 1000; 43 kDa, Affinity Biosciences), p38 (1 : 3000; 43 kDa, Affinity Biosciences), p-IκBα (1 : 1000; 39 kDa, Affinity Biosciences), Caspase1 (1 : 1000; 45 kDa, Affinity Biosciences), COX2 (1 : 1000; 72 kDa, Affinity Biosciences), iNOS (1 : 500; 130 kDa, Affinity Biosciences), and α-Tubulin (1 : 5000; ProMab). Subsequently, the blots were probed with horseradish peroxidase- (HRP-) conjugated goat secondary antibody against rabbit IgG (1 : 80000; Affinity Biosciences). Quantitative analysis of the protein bands was performed with Image-Pro Plus 5.1 software (Media Cybernetics, Inc., Bethesda).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!