Yeast whole-cell lysates, the fractions from the glycerol gradient sedimentation, and the coimmunoprecipitated proteins were resuspended in SDS sample buffer, resolved on 8 or 10% SDS-PAGE (Tris-glycine running buffer), transferred to nitrocellulose membranes (Ambion) and incubated with anti-CBP (Millipore), anti-GFP (Sigma-Aldrich), anti-uL18 (gift from Dr Cleslei F. Zanelli, UNESP), anti-Nog1 (gift from Dr John L. Woolford Jr., Carnegie Mellon University) and anti-Pgk1 (Abcam). Secondary antibodies conjugated to IR700dye (anti-rabbit IgG, LI-COR) or IR800dye (anti-mouse IgG, LI-COR) were employed, and near-infrared Western blot detection was carried out using ChemiDoc MP Imaging System (BioRad) or Odyssey equipment (LI-COR). Images were processed using Image Studio™ Lite (ver. 5.2) software (LI-COR).
Anti rabbit igg
Manufactured by LI COR
Sourced in United States
The Anti-rabbit IgG is a secondary antibody used in various immunoassay techniques to detect and quantify rabbit primary antibodies. It binds specifically to the Fc region of rabbit immunoglobulin G (IgG) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg, by LI COR (5 mentions)
Anti β actin, by Merck Group (3 mentions)
Odyssey clx, by LI COR (2 mentions)
β actin, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Anti pgk1, by Abcam (1 mentions)
Odyssey equipment, by LI COR (1 mentions)
Image studio lite ver 5, by LI COR (1 mentions)
Anti cbp, by Merck Group (1 mentions)
Anti gfp, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Irdye conjugated anti mouse igg, by LI COR (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Mouse anti γh2ax, by Merck Group (1 mentions)
Anti pgc 1α antibody, by Santa Cruz Biotechnology (1 mentions)
Anti sirt3, by Cell Signaling Technology (1 mentions)
Mouse anti nitrotyrosine, by Merck Group (1 mentions)
13 protocols using anti rabbit igg
1
Immunoblot Analysis of Yeast Complexes
2
Western Blot Analysis of Bacterial Proteins
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Bacterial whole-cell lysate preparation and trichloroacetic acid precipitation of supernatant fractions from mid-exponential cultures was performed as previously described (24 (link), 104 (link)). Indicated fractions were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and proteins of interest were probed using polyclonal rabbit anti-His6 (1:2,000; Invitrogen, Waltham, MA) and monoclonal mouse anti-RNAP (1:3,500; BioLegend, San Diego, CA). IRDye-conjugated anti-mouse IgG and anti-rabbit IgG were used as secondary antibodies (1:5,000 for each; LI-COR Biosciences, Lincoln, NE), and blots were visualized with the Odyssey CLx imaging system (LI-COR Biosciences).
3
Western Blotting Analysis of Oxidative Stress Markers
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The Western blotting was conducted as described earlier (Huang et al., 2018 (link)). Briefly, the ischemic cortex was collected at different time after reperfusion, homogenized in tissue lysing buffer. Proteins were separated with 8%–12% SDS-polyacrylamide gel and transferred to nitrification cellulose membranes with glycine transfer buffer. The membrane was sealed with 5% skim milk and protein was detected with primary antibody, including mouse anti-nitrotyrosine (#05233, Millipore, Darmstadt, Germany), mouse anti-γH2. AX (#ab2893, Abcam, Cambridge, United Kingdom), rabbit anti-4-hydroxynonenal (#ab46545, Abcam, Cambridge, United Kingdom), anti-SIRT3 (#2627, Cell Signaling Technology, Danvers, United States), anti-PGC-1α antibody (#SC13067, Santa Cruz, Dallas, United States), and mouse anti-GAPDH (#ab9484, Millipore, Darmstadt, Germany), anti-β-Actin (#A5441, Sigma, Darmstadt, Germany). After incubating overnight at 4°C, the membrane was incubated with secondary antibodies including anti-mouse IgG or anti-rabbit IgG (Li-Cor Bioscience, Lincoln, NE, United States). The protein bands were visualized using the Odyssey Infrared Imaging System (Li-Cor Bioscience). After the images were collected, the protein expression was finally determined using ImageJ Launcher software (National Institutes of Health, Bethesda, MD, United States) and normalized to a loading control GAPDH.
4
Western Blot Analysis of EV Proteins
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Cells or NK-EVs were lysed with NP40 Cell Lysis Buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Cell lysates were incubated with Loading Dye (LI-COR) for 30 min under non-reducing conditions at 37 °C to detect CD63 as previously described [29 (link)] or with Loading Dye and 2-mercaptoethenol for 5 min at 95 °C to detect other proteins. Proteins were loaded onto NuPAGE gels (Invitrogen), transferred to membranes, and the membranes were incubated with 5% skim milk in TBS-T for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies, anti-CD81, CD63 (Cosmo Bio, clone 12C4, 8A12, respectively), cytochrome c, granzyme B (Biolegend, clone 7H8.2C12, O94E6, respectively), β-actin, granzyme H (Cell Signaling Technology, catalog 4967, 18268, respectively), and α-tubulin (Santa Cruz, catalog sc-23948). Secondary antibodies, IRDye 680RD-conjugated anti-mouse IgG, or anti-rabbit IgG (LI-COR) were used at a 1:5000 dilution. The membranes were exposed to ODYSSEY CLx (LI-COR).
5
Western Blot Analysis of CSF and Serum Proteins
6
Antibody Panel for Phosphorylation Analysis
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Primary antibodies including anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-4EBP1 (Ser65), anti-4EBP1, anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-rpS6 (Ser240/244), anti-phospho-rpS6 (Ser235/236), anti-rpS6, anti-p-p53 (Ser15), p53, anti-phospho-IGF-1Rβ (T1135/36/1150/51), IGF-1Rβ and anti-MYC antibodies were purchased from Cell Signaling Technology (Danfoss, MA, USA). The anti-Rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-S6K1 antibody was purchased from Abcam (Cambridge, UK). Anti-β-actin, anti-FLAG (M2) and anti-p62/SQSTM1 antibodies were purchased from Sigma (Saint Louis, MO, USA), anti-LC3 antibody was purchased from Medical & Biological Laboratories (Nagoya, Aichi, Japan). Secondary antibodies used in western blotting including IRDye 800CW Donkey anti-Mouse and anti-Rabbit IgG were purchased from Li-COR (Lincoln, NE, USA). Secondary antibodies used in immunofluorescence including AlexaFluor-555-conjugated goat anti-mouse and AlexaFluor-555-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Burlingame, CA, USA).
7
Quantification and Western Blot Analysis
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Protein samples from organoids were lysed in RIPA lysis buffer55 (link) with protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Sigma) and quantified using a Pierce BCA protein assay kit (Thermo Fisher). Following SDS–PAGE and transfer to PVDF membranes (Bio-Rad, 1704273), membranes were blocked in Tris-buffered saline containing 5% bovine serum albumin (BSA) and 0.1% Tween 20 (TBS-T) for 1 h before incubation with primary antibody (CYP3A5, ab108624, Abcam, 1:1,000; GAPDH, cs2118, Cell Signaling, 1:1,000) overnight at 4 °C. After washing three times in TBS-T and incubating with species-corresponding secondary antibodies (anti-mouse IgG, LI-COR, 1:10,000; anti-rabbit IgG, LI-COR, 1:10,000), membranes were visualized with an ODYSSEY CLx (LI-COR) imaging system.
8
Immunoblotting of Fibrotic Markers
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DMEM was purchased from Gibco Invitrogen Corporation (Carlsbad, CA, USA). STZ was obtained from Sigma (Saint Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (BI, Beit HaEmek, Israel). Mouse and rat enzyme-linked immunosorbent assay (ELISA) kits were obtained from Elabscience Biotechnology (Wuhan, China). Antibodies were purchased from the following sources: anti-FN antibody, anti-TGF-β1 antibody, and anti-CTGF antibody were from Abcam (Cambridge, UK); anti-p-Akt1/2, anti-Akt1/2, anti-NF-κB p-p65, anti-p38, and anti p-p38 antibody were from Cell Signaling Technology, Inc. (Beverly, MA, USA); anti-α-SMA antibody was purchased from Gene Tex Inc. (Alton Parkway Irvine, CA, USA); and anti-α-tubulin monoclonal antibody was from Proteintech Group, Inc. (Chicago, USA). IRDye 680LT goat anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG were obtained from LI-COR Biosciences (Lincoln, NE, USA).
9
Protein Extraction and Immunoblotting
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Cells and tissues were lysed in RIPA lysis buffer (Thermo Fisher, 89 900) containing PMSF (Thermo Fisher, 36 978) and protease inhibitors. Total protein lysates were boiled with loading sample buffer containing 10% SDS‐PAGE. Subsequently, separated proteins were transferred onto PVDF membranes. PVDF membrane blots were blocked in 10% skimmed milk for 1 h at room temperature, washed in Tris‐buffered saline with Tween 20 (TBS‐T), and incubated overnight at 4 °C with rabbit anti‐METTL3 (Proteintech, 15073‐1‐AP), rabbit anti‐PDGFRα (CST, 3174), and mouse anti‐β‐ACTIN (Sigma‐Aldrich, A3854). Anti‐rabbit IgG (LI‐COR, 926–68071) was used as the second antibody for METTL3 and PDGFRα. Anti‐mouse IgG (LI‐COR, 926–32210) was used as the secondary antibody for β‐ACTIN.
10
Western Blot Analysis of BAFF in Lung Tissue
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Lung lobes were homogenized in RIPA lysis buffer with a Polytron homogenizer (Kinematica, Luzern, Switzerland). Total protein concentrations of pulmonary macrophages and lung lysates were determined with the DC protein assay (Biorad, Mississauga, Ontario, Canada). Lysates were resuspended in loading buffer and 15 μg (macrophage) and 40 μg (lung) of proteins were loaded per well. SDS‐PAGE was performed using 10% acrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked for 1 h (5% fat‐free milk, 0.5% Tween‐20 in PBS) and incubated overnight with rabbit IgG anti‐mouse BAFF (1:1000; Abcam, Cambridge, United Kingdom) and goat IgG anti‐mouse β‐actin (1:4000; Abcam) at 4°C. Anti‐rabbit IgG coupled to IRdye 800 nm and anti‐goat IgG coupled to IRdye 700 nm were used as detection antibodies (1:5000, LI‐COR Bioscience, Lincoln, NE). Staining was visualized with the Odyssey CLx Imager (LI‐COR Bioscience). Band intensity was quantified with the Image Studio Lite software (LI‐COR Bioscience) and reported as BAFF intensity over β‐actin intensity for every sample.
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