After washing with PBS, the cells were lysed with RIPA buffer (Beyotime). The supernatants of the cell lysates were collected after centrifugation and quantified with the BCA protein assay. Equal amounts of proteins were separated on SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk buffer for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. Then, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, CWBIO) for 1 h at room temperature. The membranes were then visualized with enhanced chemiluminescence (ECL, Advansto). GAPDH was used as an internal control for protein loading. The target protein’s relative expression was shown as a ratio of target protein/GAPDH.
Goat anti rabbit igg
Manufactured by CWBIO
Sourced in China
Goat anti-rabbit IgG is a secondary antibody used in immunoassays to detect and quantify rabbit primary antibodies. It is produced by immunizing goats with purified rabbit immunoglobulin G (IgG) and can bind to and label rabbit IgG molecules.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by CWBIO (5 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Occludin, by Abcam (1 mentions)
β actin, by CWBIO (1 mentions)
Nlrp3, by Abcam (1 mentions)
Caspase 1, by Affinity Biosciences (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Gapdh, by GeneTex (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Gr68497 2, by Merck Group (1 mentions)
Anti cox 4 antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti parp antibody, by Cell Signaling Technology (1 mentions)
12 protocols using goat anti rabbit igg
1
Western Blot Analysis of Target Proteins
2
Immunohistochemical Staining of Basal Forebrain and Hippocampus
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Basal forebrain and hippocampal slices were rinsed in PBS and incubated in 3% hydrogen peroxide. After blocking in 10% bovine serum albumin (BSA, Sigma, USA), rabbit anti-rat P75 antibody (1:5 000, Sigma, USA) was applied to basal forebrain slices, while rabbit anti-rat synaptophysin antibody (1:100, Sigma, USA) was applied to hippocampal slices. After 4°C overnight incubation, brain slices were treated with goat anti-rabbit IgG (Cwbio, China) for 2-h incubation at room temperature. Horseradish peroxidase (HRP) was then applied to tissue slices for 10-min incubation at room temperature. The signal was visualized by DAB chromogenic substrate (Maixin, China). After gentle rinsing, dehydration, and mounting, staining images were captured using a light-field microscope.
3
Metformin Modulates Gut Inflammation
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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).
4
Western Blot Analysis of Testicular Proteins
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Total protein was extracted from the frozen testicular tissues into RIPA buffer (Cat. No. CW2333S, CWBIO) that contained a phosphatase inhibitor cocktail (100×, Cat. No. CW2383S, CWBIO) and a protease inhibitor cocktail (100×, Cat. No. CW2200S, CWBIO). The protein concentration was quantified by the Pierce BCA Protein Assay Kit (Cat. No. 23227, Thermo Fisher Scientific, Waltham, MA, USA). Protein lysates (20 μg) were separated with 10% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, IPVH00010, Millipore). Then, the membranes were blocked in a 5% (w/v) nonfat milk solution and incubated with antibodies for UCLH1 (dilution: 1:1000), CYP11A1 (dilution: 1:1000, Cat. No. GTX56293, Genetex, Irvine, CA, USA), SOX9 (dilution: 1:500), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; dilution: 1:4000, Cat. No. T0004, Affinity, Cincinnati, OH, USA) overnight at 4°C. Finally, the membrane was incubated with the appropriate secondary antibody (goat anti-rabbit IgG, 1:5000, CW0103S, CWBIO; goat anti-mouse IgG, 1:5000, CW0102S, CWBIO) and visualized with an enhancer (ChemiDoc Imaging System, BIO-RAD Laboratories, Hercules, CA, USA).
5
Protein Expression in Optical Tectum
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Animals were anesthetized and the tecta were dissected. The optical tecta (approximately 30–50 brains for each group) were homogenized in radioimmune precipitation assay (RIPA) buffer with a protease inhibitor cocktail (Sigma Aldrich) and phenylmethylsulfonyl fluoride (PMSF, Solarbio) at 4°C. Protein concentrations were measured by BCA assay using a Nanodrop (Thermo Scientific, 2000c). Protein homogenates were separated by SDS-PAGE (Bio-Rad). PVDF membranes were blocked in 4% non-fat milk for 1 h with TBS buffer containing 0.1% Tween-20 (Sigma Aldrich) (TBST) and incubated with primary antibodies overnight at 4°C. Antibodies were diluted in 1% non-fat milk. We used the following antibodies: anti-acetylation H3K9 (1:2000, Rabbit, Abcam, ab10812), anti-GAD67 (1:20000, Rabbit, Sigma, G5163), anti-VGAT (1:2000, Rabbit, ABclonal, A3129), and anti-GAPDH (1:10000, Rabbit, Millipore, GR68497-2). Blots were rinsed with TBST and incubated with the following horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature: goat anti-rabbit IgG (1:2000, CWbiotech, CW0103) and goat anti-mouse IgG (1:2000, CWbiotech, CW0102). Bands were visualized using ECL chemiluminescent (1:1, Pierce).
6
Molecular Pathways in Cancer Signaling
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SNS-032 (#S1145) and CDDP (#S1166) were purchased from Selleck Chemicals (Shanghai, China). The antibodies against RNA Pol II (#A300-653A), p-RNA Pol II (S5) (#A304-408A), and p-RNA Pol II (S2) (#A300-654A) were obtained from Bethyl Laboratories (Montgomery, TX). The anti-p-RNA Pol II (S7) (#04-1570) was purchased from EMD Millipore (Billerica, MA). The anti-CDK7 antibody (#2916), anti-CDK9 antibody (#2316), anti-Caspase-3 antibody (#9665), anti-PARP antibody (#9532), anti-Cleaved Caspase-3 antibody (#9664), anti-Bcl-XL antibody (#2762), anti-Bcl-w (31H4) (#2724), anti-XIAP antibody (#2042), anti-Mcl-1 antibody (#5453), anti-Cytochrome c antibody (#4272), anti-AIF antibody (#5318), anti-COX IV antibody (#4850), anti-MMP-2 antibody (#4022), and anti-GFP antibody (#2956) were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The antibody against Ki67 (#ab15580) and MMP-1 (#ab137332) were obtained from Abcam (Cambridge, UK). The anti-Actin antibody (#4700) was purchased from Sigma (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies including Goat anti-Mouse IgG (#CW0102S) and Goat anti-Rabbit IgG (#CW0103S) were bought from CWBio (Beijing, China).
7
Immunohistochemical Analysis of Aortic Biomarkers
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Aortic cross-sections were fixed with paraformaldehyde for 12 h and processed for immunostaining for VCAM-1, p-STAT3, and GATA6, followed by incubation with goat anti-rabbit IgG (CWBIO, Beijing, China). After washing with PBS, color was developed with diaminobenzidine (DAB) solution (CWBIO, Beijing, China), and the nuclei were counterstained with Harris hematoxylin for 15 s. Unless specified, the sections were blocked with 1% BSA plus normal horse serum or 1% BSA alone. Negative controls were performed by omitting primary antibody, and the representative pictures were shown in Supplementary data (Figure S9 ). Photographs of immunostained mouse aorta (200x magnification) were digitized and captured using an inverted bright field and phase contrast microscope (Nikon Ti-S, Japan).
8
Locust Brain Protein Analysis
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Locust brains (8–10 individuals/sample) were collected and homogenized in lysis buffer (CWBIO) containing protease inhibitor (CWBIO). The total protein content was determined using a bicinchoninic acid protein assay kit (Thermo Scientific). The extracts were reduced, denatured, and separated by gel electrophoresis on a 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated separately with specific antibodies against Piwi1, Piwi2, and Ago3 (mouse anti‐Piwi1 serum, 1:500; mouse anti‐Piwi2 serum, 1:500; and mouse anti‐Ago3 serum, 1:500, respectively), hnRNP F/H (Mouse monoclonal antibody, 1:2,000, Abcam), and U2AF65 (Mouse monoclonal antibody, 1:200, Santa Cruz Biotechnology). Tubulin was used as an endogenous control (rabbit polyclonal antibody, 1:5,000, CWBIO). Goat anti‐rabbit IgG was used as the secondary antibody (1:10,000, CWBIO). The intensities of the Western blot bands were quantified using densitometry with Quantity One software.
9
Tracking SARS-CoV-2 Protein Expression
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MARC-145 cells were infected with a MOI of 0.1, and lysed in SDS loading buffer at 8, 16, 24, 48, 72, and 96 h post infection (hpi). The proteins in cell lysis solution were separated by SDS-PAGE, and transferred onto a PVDF membrane. After blocking with 5% skimmed milk overnight at 4°C, the membranes were, respectively, incubated with anti-NSP7α and anti-NSP7β polyclonal antibodies, followed by incubation with goat-anti-rabbit IgG (CWBIO, China) conjugated horseradish peroxidase as secondary antibody. Immunodetection was performed using enhanced chemiluminescence (ECL) reagents (CWBIO, China) according to the supplier’s instruction.
10
Protein Extraction and Western Blot Analysis
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Total protein extraction was carried out by lysing cells with 1× SDS loading buffer and denatured at 95°C for 5 min. The sample volume per lane was 10 μL. The protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Billerica, USA). After being blocked and washed, the membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with the corresponding secondary antibodies. Finally, the membrane was exposed with ECL developer (CW0049M; CWbio, Beijing, China) and processed with ImageJ software (
https://imagej.nih.gov/ij/ ). The primary antibodies used were as follows: anti-GAPDH (1:1000; CWbio), anti-HDAC6 (1:1000; 12834-1AP; Proteintech), anti-acetylated tubulin (1:1000; T7451; Sigma, St Louis, USA), anti-α-tubulin (1:1000; T6199; Sigma), anti-collagen II (1:1000; ab34712; Abcam), anti-MMP13 (1:1000; Wanleibio, Shenyang, China), and anti-SOX9 (1:1000; ab182579; Abcam). The secondary antibodies were goat anti-mouse IgG (1:4000; CWbio) and goat anti-rabbit IgG (1:4000; CWbio).
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