The samples were obtained from the kidneys for immunoblotting. The tissues were homogenized in RIPA buffer (#89900. Thermo scientific. Waltham, MA, USA). Amounts of protein were measured using the BCA assay kit (Pierce, Rockford, IL, USA), according to the manufacturer’s protocol. Proteins (50 µg) were loaded and electroblotted. The blots were probed with primary antibodies against monoclonal anti-caspase-1 (Abcam) and polyclonal anti-NLRP1 (Cell signaling, Danvers, MA, USA), NLRP-3 (Abcam, Cambridge, UK), COX-2 (Cell signaling), and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The primary antibody was visualized by a secondary antibody and an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The β-actin antibody (Sigma, St. Louis, MO, USA) served as the loading control. The densitometric analysis was performed for quantitative analysis of all data.
Anti nlrp1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-NLRP1 is a primary antibody that specifically recognizes the NLRP1 protein. NLRP1 is a member of the NOD-like receptor (NLR) family and plays a role in the innate immune response.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 1, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Nlrp3, by Abcam (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Anti gsdmd n, by Abcam (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Ab214185, by Abcam (1 mentions)
Beyoecl plus substrate, by Beyotime (1 mentions)
Anti p nrf2, by Bioss Antibodies (1 mentions)
Ab137550, by Abcam (1 mentions)
P0013, by Beyotime (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
4 protocols using anti nlrp1
1
Renal Immunoblotting for Inflammasome Proteins
2
Western Blot Quantification of Inflammatory Proteins
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Total protein was extracted from mouse kidney tissue and HUVECs using RIPA lysis buffer (Beyotime, cat. no. P0013B) on ice; the obtained lysate was centrifuged at 12,000 × g at 4°C for 30 min. Protein quantification was performed by using a BCA Protein Assay Kit (Thermo Fisher, cat.no. A53225). Proteins were separated by 10% SDS‐PAGE and then transferred to an NC membrane (Pall Bio Trace, cat. no. 66485) using a Bio‐Rad Trans‐Blot Turbo (USA) system. After blocking with 5% fat‐free milk powder in TBST buffer, the membranes were incubated with the following primary antibodies overnight at 4°C: anti‐GAPDH (abcam, cat. no. ab9485), anti‐caspase‐1 (Cell Signaling Technology, cat. no. 3866), anti‐NLRP3 (Cell Signaling Technology, cat. no. 13158), anti‐NLRP1 (Cell Signaling Technology, cat. no. 4990), anti‐GSDMD‐FL (Invitrogen, cat. no. PA5‐104324) and anti‐GSDMD‐N (abcam, cat. no. ab215203). Next, the membranes were incubated with horseradish peroxidase–conjugated anti‐rabbit IgG secondary antibodies for 2 h at room temperature. Bands were detected using a ChemiDoc MP Imaging System (Bio‐Rad), and ImageJ 1.51 software was used for the quantitative analysis of all protein bands.
3
Western Blot Analysis of Neurological Proteins
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Total proteins from cultured PC12 cells or rat brain tissues were extracted using cell lysis buffer for Western blotting (P0013; Beyotime Biotech) according to the manufacturer’s instructions. Protein concentrations were analyzed using the BCA method. About 25 μg of protein from each group was boiled in loading buffer for 5 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membranes. Subsequently, membranes were blocked by incubation with 5% BSA at room temperature for 2 h, incubated with diluted primary antibodies for 1–2 h at room temperature. After washing twice with Tris-buffered saline containing Tween, membranes were incubated with diluted secondary antibodies for 2 h at room temperature and developed using BeyoECL Plus Substrates (P0018S; Beyotime Biotech). The abundance of GAPDH was used as an internal standard. Antibodies used for Western blotting were anti-TH (25859-1-AP; Proteintech), anti-NRF2 (ab137550; Abcam), anti-p-NRF2 (#bs2013R; BIOSS, Beijing, China), anti-α-syn (4179; Cell Signaling Technology, Danvers, MA, USA), anti-caspase 1 (p10; 22915-1-AP, Proteintech), anti-NLRP3 (ab214185, Abcam), and anti-NLRP1 (4990, Cell Signaling Technology).
4
Quantifying Cellular Protein Levels
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For protein quantification, SH-SY5Y cells were seeded in 6-well plates (2x10 6 cells/well) and allowed to stabilise for 24 hours. The cells were then treated as described for 2 hours. Total protein in the cell lysate was harvested in RIPA buffer (Abcam, UK) and quantified using the Bradford assay (Bio-Rad, USA). Proteins were also separated using 10% sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gel at 120V where each well was loaded with 30µg protein in Laemmli buffer (Bio-Rad, USA). The gel was blotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with Blocking One (Nacalai Tesque, Japan) for one hour at room temperature. The proteins of interest in each sample were probed using primary antibodies (anti-NLRP1, anti-caspase-1 and anti-β-actin; Cell signalling Technology, USA) at 1:1000 dilution. β-actin was used as loading control. Anti-rabbit IgG HRP-conjugated antibody (Cell Signalling Technology, Massachusetts, USA) at 1:2000 was used as secondary antibody. The Chemi-Lumi One Super enhanced chemiluminescence reagent (Nacalai Tesque, Japan) was used to detect the blots.
Blot visualization and densitometric analyses were performed using Bio-Rad Image Lab 6.0.1 software. All western blots were performed in triplicates.
Blot visualization and densitometric analyses were performed using Bio-Rad Image Lab 6.0.1 software. All western blots were performed in triplicates.
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