Anti il 1β

Manufactured by Affinity Biosciences

Sourced in United States

Anti-IL-1β is a laboratory reagent used for the detection and quantification of interleukin-1 beta (IL-1β) in biological samples. It functions as an antibody that specifically binds to IL-1β, enabling its identification and measurement.

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8 protocols using anti il 1β

Western Blot Protocol for Protein Analysis

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The protein extraction process in this study can be referred to the existing study of Yi et al. (2018) (link). Protein weighing 20–30 µg were electrophoresed on SDS polyacrylamide gels. We then transferred the proteins to membranes, which were blocked with skimmed milk (5%) for 2 h at room temperature. Incubation of primary antibodies with the proteins was stored overnight at 4°C as follows: anti-P2X7 (Affinity Biosciences, OH, United States), anti-TRPV1 (Affinity Biosciences, OH, United States), anti-IL-18 (Affinity Biosciences, OH, United States), anti-TNF-α (Affinity Biosciences, OH, United States), anti-IL-6 (Affinity Biosciences, OH, United States), anti-IL-1β (Affinity Biosciences, OH, United States), or anti-GAPDH (Affinity Biosciences, OH, United States).
The membranes were rinsed with TBST for 10 min for triplicates and incubated for 2 h with the secondary antibodies (Boster Biological Technology Co. Ltd.). ECL chemiluminescence detection and development with a gel imaging system were performed finally. Results analysis for this section was realized via ImageJ.

Long L., Zhong W., Guo L., Ji J, & Nie H. (2022). Effect of Bufalin-PLGA Microspheres in the Alleviation of Neuropathic Pain via the CCI Model. Frontiers in Pharmacology, 13, 910885.

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Western Blot Analysis of NLRP3 Inflammasome

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Cell and tissue proteins were extracted as previously described (19 (link)), and bicinchoninic acid method was used to determine the protein concentration. Nitrocellulose membranes were incubated with primary antibodies (anti-NLRP3, anti-IL-18, GAPDH, Proteintech Group, Inc., Chicago, IL,USA; anti-GSDMD, Abbexa Ltd, Cambridge, United Kingdom; anti-caspase-1, anti-IL-1β, anti-cleaved caspase-1, anti-cleaved IL-1β, Affinity Biosciences, Cincinnati, OH, USA; anti-caspase-11 p20, Santa Cruz Biotechnology, Inc.Dallas, Texas, USA) at 4 °C overnight. The membranes were washed with 1% TBST before and after incubation with goat anti-rabbit IgG secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) or goat anti-mouse IgG secondary antibody (LI-COR Biotechnology, Lincoln, NE, USA) for 1 h at room temperature. Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA) was used to analysis protein expression as previously described (19 (link)).

Liu W., Yang D., Shi J., Wen P., Zhang J., Wang Z., Hu B., Shi X., Cao S., Guo W, & Zhang S. (2021). Caspase-1 Inhibitor Reduces Pyroptosis Induced by Brain Death in Kidney. Frontiers in Surgery, 8, 760989.

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Anti-inflammatory and Apoptosis Modulation

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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). BRB, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), HAuCl₄. 3H₂O (≥99.9% trace metals basis), BSA, Triton X-100 and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (USA). RIPA lysis buffer, phenylmethanesulfonyl fluoride, hematoxylin-eosin (HE) staining kit and Nissl staining solution (methylene blue) were obtained from Solarbio (China). The antibodies to anti-CD86, anti-CD206, anti-Cleaved Caspase-3 and anti-TNF-α were purchased from Cell Signaling Technology (USA). Anti-Bax, anti-Bcl-2, anti-IL-1β and anti-IL-6 were purchased from Affinity (USA). Anti-β-Tubulin, anti-GAPDH, HRP Affinipure Goat Anti-Mouse IgG and HRP Affinipure Goat Anti-Rabbit IgG were purchased from Proteintech (USA). The Alexa Fluor^®568 goat anti-mouse/rabbit IgG and Alexa Fluor^®488 goat anti-mouse/rabbit IgG were purchased from Invitrogen (USA). 4,6-dimethyl-2-phenylindole (DAPI) was purchased from Abcam (UK). RAW 264.7 cells and VSC 4.1 cells were obtained from the American type culture collection.

Zhou Z., Li D., Fan X., Yuan Y., Wang H., Wang D, & Mei X. (2021). Gold nanoclusters conjugated berberine reduce inflammation and alleviate neuronal apoptosis by mediating M2 polarization for spinal cord injury repair. Regenerative Biomaterials, 9, rbab072.

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Kidney Nlrp3 Inflammasome Protein Analysis

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The kidney tissues were homogenized and the supernatant was collected after centrifugation at 12,000 ×g at 4°C for 20 min [23 (link)]. We separated the lysates on 10% polyacrylamide gels before immunoblotting using anti-Nlrp3 (AdipoGen company, San Diego, CA), anti-ASC (AdipoGen company, San Diego, CA), anti-CHOP (Cell Signaling Technology, USA), anti-caspase-12 (Cell Signaling Technology, USA), anti-IL-1β (Affinity Biosciences, USA), and anti-IL-18 (Affinity Biosciences, USA) antibodies at a dilution of 1 : 500. The expression levels of CHOP, caspase-12, ASC, Nlrp3, IL-1β, and IL-18 were analyzed using an ECL advance system (Amersham, Little Chalfont, UK). The relative protein expression levels were determined by normalization to β-actin. Serum IL-1β and IL-18 were measured with ELISA kits (RayBiotech, Norcross, GA) according to the manufacturer's instruction.

Guo H., Li H., Ling L., Gu Y, & Ding W. (2016). Endoplasmic Reticulum Chaperon Tauroursodeoxycholic Acid Attenuates Aldosterone-Infused Renal Injury. Mediators of Inflammation, 2016, 4387031.

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Rat Kidney NLRP3 Inflammasome Activation

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We lysed and Western blotted rat renal tissue as described previously [17 (link)]. We separated lysates on 10% polyacrylamide gels, before immunoblotting using anti- NLRP3 (adipoGen company, San Diego, CA), anti-ASC (adipoGen company, San Diego, CA) antibody, anti-IL-1β (Affinity Biosciences, USA) and anti-IL-18 (Affinity Biosciences, USA) antibody at a dilution of 1:500. We used a semi-quantitative analysis using densitometry, and used β-actin to standardize the amount of protein loaded on the blots.

Ding W., Xu C., Wang B, & Zhang M. (2015). Rotenone Attenuates Renal Injury in Aldosterone-Infused Rats by Inhibiting Oxidative Stress, Mitochondrial Dysfunction, and Inflammasome Activation. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 21, 3136-3143.

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Neuroinflammatory Signaling Pathway Analysis

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The anti-P2X7R antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p38, anti-p-p38, anti-IL-1β, anti-IL-6, anti-TNF-a, anti-IL-10 and anti-Synaptotagmin 1 antibodies were obtained from Affinity Biosciences (Cincinnati, OH, USA). The anti-Iba1 antibody was obtained from Wako (Wako Pure Chemical Industries, Ltd). An anti-GAPDH was obtained from Bioworld. The anti-BDNF antibody and the P2X7R-specific agonist BzATP were obtained from Abcam (Cambridge Science Park, Cambridge, UK). An FD Rapid GolgiStain Kit was obtained from FD Neurotechnologies, Inc. (Guilford, MD, USA).

Wu Q., Yue J., Lin L., Yu X., Zhou Y., Ying X., Chen X., Tu W., Lou X., Yang G., Zhou K, & Jiang S. (2021). Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression. Molecular Pain, 17, 1744806921997654.

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Protein Expression Analysis in Cells

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Proteins from cells and lung tissues and the immunoprecipitated proteins were used. After protein concentration determination by NanoPhotometer NP80^® Implen (Bavaria, Munich, Germany), 20 μg of total protein was separated using a 12% SDS-PAGE gel. The primary antibodies included anti-DEK (#29812S, CST), anti-MFN1 (#57602, Abcam), anti-DRP1 (#8570S, CST), anti-sequestosome 1 (SQSTM1)/p62 (#211324, Abcam), anti-LC3B (#63817, Abcam), anti-Cytochrome c oxidase IV (COX IV, #16056, Abcam), anti-Cytochrome c (#133504, Abcam), anti-NLRP3 (#15101, CST), anti-caspase-1 (#AB1871, Sigma, St. Louis, MO, USA), anti-IL-1β (#AF5103, Affinity, USA), anti-Cleaved caspase-3 (#9664, CST), anti-Bax (#182858, Abcam), anti-Bcl-2 (#17509, Abcam), anti-PINK1 (#DF7742, Affinity, USA), anti-Parkin (#A0968, ABclonal, USA), anti-MnSOD (#AF5144, Affinity, USA), ATAD3A (#A8230, ABclonal), and anti-GAPDH (#5174, CST). The secondary antibodies were the HRP-goat anti-rabbit antibody (#5151, CST) and the HRP-anti-mouse antibody (#5257, CST). The internal controls were COX IV and GAPDH. The gray values of protein bands were calculated by Quantity One software (BioRad, Hercules, CA, USA).

Bai Q., Liu R., Quan C., Han X., Wang D., Wang C., Wang Z., Li L., Li L., Piao H., Song Y, & Yan G. (2024). DEK deficiency suppresses mitophagy to protect against house dust mite-induced asthma. Frontiers in Immunology, 14, 1289774.

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Quantifying Ovarian Protein Levels

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100 mg of ovarian tissue from each of the four groups was homogenized with RIPA lysis buffer for 30 minutes on ice, and the total protein concentration was measured using the bicinchoninic acid assay. Separate the protein sample (50 μg) using SDS-PAGE and electrotransfer to a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with the following antibodies: anti-MAPK1 (CST, USA), anti-CXCL8 (Affinity, USA), anti-IL-6 (Affinity, USA), anti-IL-1β (Affinity, United States), and anti-GADPH (Affinity, United States). The membrane was further incubated with the secondary antibody; ECL-Plus chemiluminescence was used to detect immunoreactive bands.

Ma J.X., Ye M., Ma K., Zhou K., Zhang Y., Wang X, & Tong H. (2021). Network Pharmacology-Based Strategy for Predicting Active Ingredients and Potential Targets of Coptis chinensis Franchin Polycystic Ovary Syndrome. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 6651307.

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