Histones were isolated from log phase of WT strains and mutants using total histone extraction kit (Epigentek, USA), then separated by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF membranes (110 V, 3.5 h). The membranes were blocked in 5% nonfat milk and incubated with anti-HA (1:300, Millipore) primary antibody overnight at 4°C. Following three washes, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:800, Millipore) for 1 h at 37°C. Finally, blots were developed with Western Blotting Detection Reagent (Engreen, China) according to the product instructions.
Hrp conjugated goat anti rabbit igg antibody
Manufactured by Merck Group
Sourced in United States
The HRP-conjugated goat anti-rabbit IgG antibody is a laboratory reagent used in various immunoassay techniques. It consists of a goat-derived antibody that specifically binds to rabbit immunoglobulin G (IgG) molecules, with a horseradish peroxidase (HRP) enzyme conjugated to it. This antibody-enzyme conjugate can be utilized as a detection tool in assays where the presence or quantity of rabbit IgG needs to be determined.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg antibody, by Merck Group (3 mentions)
4 chloro 1 naphthol, by Merck Group (2 mentions)
Bio1d software, by Vilber (2 mentions)
Anti ha, by Merck Group (1 mentions)
Total histone extraction kit, by Epigentek (1 mentions)
Versadoc 4000 mp, by Bio-Rad (1 mentions)
Oneblock western cl blocking buffer, by Genesee Scientific (1 mentions)
Clarity ecl substrate, by Bio-Rad (1 mentions)
Mouse anti gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Pageruler plus, by Thermo Fisher Scientific (1 mentions)
Hrp labeled goat anti mouse igg, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Blue loading buffer, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Hrp conjugated goat anti rat igg antibody, by Merck Group (1 mentions)
F4 80, by Abcam (1 mentions)
Biozero software, by Keyence (1 mentions)
Seeblue plus2, by Thermo Fisher Scientific (1 mentions)
Hrp labeled donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
20 protocols using hrp conjugated goat anti rabbit igg antibody
1
WT Histone Extraction and Detection
2
Western Blot Analysis of FGF21 Protein
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Samples from each group were collected in RIPA lysis buffer (50 mM Tris-Cl [pH 7.4], 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 150 mM NaCl), and a protease inhibitor tablet was added (Beyotime Sciences, USA). The samples were centrifuged at 12,000 rpm at 4°C, and protein concentration was determined using a BCA protein assay (CwBio Sciences, CN). Lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membrane was probed with β-actin horseradish peroxidase (HRP) (Proteintech, CN) and blocked with 5% BSA before incubation with the primary rabbit anti-mouse FGF21 antibody (Abcam, UK). An HRP-conjugated goat anti-rabbit IgG antibody (Millipore) was applied, and after incubation, the membrane was developed with an Amersham-enhanced chemiluminescence western blotting detection system (Bio-Rad, Versa Doc 4000MP, USA).
3
Western Blot Analysis of AGA Enzyme Expression
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Cultures were harvested with PBS/EDTA and cell pellets were extracted as described above for the AGA enzyme assay. The protein content of the extracts was determined using a BCA Assay Kit (Pierce). Extracts (10 μg per lane) and molecular weight markers (Page Ruler Plus, Thermo Scientific, Waltham, MA) were separated by SDS/PAGE using a 4–12% Bis-Tris polyacrylamide gel, transferred to a 0.45 PVDF membrane, and probed with rabbit anti-AGA antibody (a gift from Transkaryotic Therapies, Inc., Cambridge, MA) at a dilution of 1:2000 in OneBlock™ Western-CL Blocking Buffer (Genesee Scientific, El Cajon, CA). Protein bands were labeled using HRP-conjugated goat anti-rabbit IgG antibody (Millipore, Temecula, CA) and they were visualized with Clarity ECL substrate (Bio-Rad, Hercules, California). Chemiluminescence was detected with an Amersham™ 600 digital imager (GE Healthcare Life Sciences, Pittsburgh, PA). HEK-293 cells transfected with a plasmid expressing human AGA (a gift from Dr. Roscoe Brady, National Institutes of Health, Bethesda, MD) were used as positive controls.
For loading controls, blots were reprobed with mouse anti-GAPDH antibody (Thermo Fisher Scientific) at 1:2000 in OneBlock™. Protein bands were detected with HRP-labeled goat anti-mouse IgG (Millipore) followed by development with Clarity ECL substrate and digital imaging with an Amersham™ 600 digital imaging system.
For loading controls, blots were reprobed with mouse anti-GAPDH antibody (Thermo Fisher Scientific) at 1:2000 in OneBlock™. Protein bands were detected with HRP-labeled goat anti-mouse IgG (Millipore) followed by development with Clarity ECL substrate and digital imaging with an Amersham™ 600 digital imaging system.
4
Quantifying FcRn Expression in Mouse Tissues
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Various human FcRn transgenic mouse tissue extracts (muscle, lung, kidney, heart, and GI) were separated on the SDS-PAGE gel and transferred onto the PVDF membrane as described earlier. The membrane was blocked with 5% milk in TBST for 1 h at room temperature. After blocking, the membrane was probed with 1 µg/mL of rabbit anti-hFcRn antibody (Abcam) or 6.8 µg/mL of 7E9 (mouse anti-hFcRn mAb) in TBST with 5% milk. The membrane was washed 4 times with TBST and probed with 0.2 µg/mL of HRP-conjugated goat anti-rabbit IgG antibody (Millipore) in TBST with 5% milk or 1 µg/mL of HRP-conjugated goat anti-mouse IgG antibody (Millipore) in TBST with 5% milk. After 90 min under a secondary antibody incubation, the membrane was washed 5 times with TBST and incubated in the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5 min. Lastly, the membrane was analyzed with the use of a ChemiDOC XRS system.
5
Western Blot Analysis of Exosome Treatments
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NHKs were harvested at 24 or 48 h after Exo treatment and lysed in a radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). The detailed methods have been described previously [63 (link)]. The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were mixed with 3× blue loading buffer (Cell Signaling Technology, Danvers, MA, USA) and heated for 3 min at 95 °C. Samples were subjected to gel electrophoresis, electro-transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked with a 5% (w/v) bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (PBST) for 1 h at room temperature. The PVDF membranes were incubated with the primary antibodies (Table 2 ). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:3000; Millipore, Billerica, MA, USA) and HRP-conjugated goat anti-mouse IgG antibody (1:3000; Millipore). Images were obtained using a chemiluminescence imaging system (WSE-6100; ATTO, Tokyo, Japan) and the optical density of the bands was measured using the Image J software (Version 1.53, NIH, Bethesda, MD, USA). Protein expression was normalized to that of β-actin; the ratio of Exo-treated cells to DPBS-treated control cells was calculated (DPBS-treated control cells = 1.0).
6
Analyzing Macrophage and Nox4 Levels in Kidneys
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Immunoperoxidase staining was performed as previously described [17] (link). In brief, macrophage infiltration was analyzed using a monoclonal antibody for murine monocyte/macrophage (F4/80, Abcam), followed by HRP-conjugated goat anti-rat IgG antibody (Millipore). The number of F4/80-positive cells was calculated in 10 glomeruli and intestitia per animal, and the mean number of F4/80 positive cells per glomerulus and interstitial tissue (number per mm2) was used for the estimation.
NADPH oxidase 4 (Nox4) immunoperoxidase staining was performed as described previously [19] (link). Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore). The proportion of the area stained with Nox4 antibody of the total area was calculated using BIOZERO software (Keyence). To quantify the proportional area of staining, 10 views of the renal cortex were randomly selected in each slide.
NADPH oxidase 4 (Nox4) immunoperoxidase staining was performed as described previously [19] (link). Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore). The proportion of the area stained with Nox4 antibody of the total area was calculated using BIOZERO software (Keyence). To quantify the proportional area of staining, 10 views of the renal cortex were randomly selected in each slide.
7
Quantitative Western Blotting of AGA Enzyme
8
Detecting TAGE Proteins in SH-SY5Y Cells
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After treatment with GA, SH-SY5Y cells were collected and homogenized with lysis buffer [7M urea, 2M thiourea, 4% CHAPS, 30 mM Tris, supplemented with 1% protease inhibitor cocktail], then the homogenate was centrifuged at 30,000× g at 4 °C for 30 min and the supernatant collected. TAGE proteins were detected by 2-DE as described previously [18 (link)]. Immobiline Dry Strips (18 cm, pH 4–7; GE Healthcare, Tokyo, Japan) were rehydrated with rehydration buffer at room temperature overnight. The first-dimension (isoelectric focusing; IEF) was run using Cool PhoreStar IPG-IEF (Anatech, Tokyo, Japan). After 2-DE of 10% SDS-polyacrylamide gel electrophoresis was run, the proteins were transferred to a nitrocellulose membrane (GE Healthcare, Tokyo, Japan) and TAGE proteins were detected using primary rabbit anti-TAGE antibody (1:500), secondary HRP-conjugated goat anti-rabbit IgG antibody (Sigma Aldrich), and ECL western blotting detection reagents (GE Healthcare, Tokyo, Japan).
9
RhoA Activation Assay and Apoptosis Analysis
10
Western Blot Analysis of SR-A Expression
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RAW264.7 cells were lysed with 100 μL radio immuno-precipitation assay lysis buffer (Sigma-Aldrich). Protein concentrations were measured with bicinchoninic acid assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a poly (vinylidene fluoride) membrane. The membrane was blocked with PBS containing 5% nonfat milk for 2 h at room temperature, then incubated overnight at 4°C with an anti-SR-A antibody (1:1,000; Abcam), or an anti-β-actin antibody (1:1,000; Cell Signaling, Danvers, MA, USA). The membrane was then washed three times for 5 min with 15 mL Tris-buffered saline Tween-20 and incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution; Sigma-Aldrich) for 1 h at room temperature. After washing, 1 mL chemiluminescent substrate (Thermo Fisher Scientific) was added to the membrane. The signal was detected and quantified with an enhanced chemiluminescence system (ImageQuant LAS-4000 MINI; GE, Fairfield, CT, USA). All samples were normalized to β-actin. Data were represented as mean ± standard error of the mean (SEM).
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