Both Western blot and co-immunoprecipitation were performed as described previously [25 (link)]. For whole-cell extracts, cells were lysed with 20 mM Tris·HCl, pH 8.0, 100 mM NaCl, 0.5% NP-40, and 1 mM EDTA (NETN buffer) supplemented with Micrococcal Nuclease (1:5000, M0247S, NEB) on ice for 15 min. Cell lysates were boiled with 5× SDS loading buffer, separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunoblotted with indicated antibodies. For co-IP experiments, cells were lysed with NETN buffer for 15 min on ice, followed by centrifugation at 12,000 rpm for 10 min at 4 °C. Supernatants were then transferred into new Eppendorf tubes and incubated with either Flag beads (L00425, GenScript) for 4 h or Protein A/G plus agarose beads (sc-2003, Santa Cruz) conjugated with indicated antibodies overnight at 4 °C with rotation. Beads were subsequently washed three times with NETN buffer and boiled with 1× SDS loading buffer. To avoid the noise of light/heavy chains, the secondary antibodies used for the immunoprecipitation assays including mouse anti-rabbit IgG LCS (A25022, 1:5000) and goat anti-mouse IgG LCS (A25012, 1:5000) were obtained from Abbkine.
A25022
Manufactured by Abbkine
Sourced in China, United States
The A25022 is a laboratory instrument designed for performing spectroscopic analysis. It measures the absorption or emission spectra of samples, providing information about their chemical composition and structure.
Automatically generated - may contain errors
Lab products found in correlation
A25012, by Abbkine (2 mentions)
Micrococcal nuclease, by New England Biolabs (1 mentions)
Mouse anti rabbit igg lcs, by Abbkine (1 mentions)
Flag beads, by GenScript (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin, by Beyotime (1 mentions)
Anti flag, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Protein a g plus agarose sc 2003, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab58632, by Abcam (1 mentions)
Ab150120, by Abcam (1 mentions)
Hrp labeled goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Hrp labeled goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
5 protocols using a25022
1
Protein Interaction Verification Techniques
2
Western Blot Analysis of FTO and NOD1
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Cellular lysates were generated by using 1×SDS-PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Beyotime; P0012S), then subjected to SDS-PAGE (8%) gel and transferred to PVDF (Millipore; ISEQ00010) membranes by semidry blotting (Bio-Rad Trans Blot Turbo System). The membranes were blocked with 5% BSA. Protein was blotted with different antibodies. The antibody against FTO was diluted at 1: 500 (Beyotime; AF6936); The antibody against NOD1 was diluted at 1: 1000 (Customized by Genscript); The antibody against NOD1 of human was diluted at 1: 1000 (Absin; abs115515); Anti-Flag (Beyotime; AF519) and anti-Tubulin (Beyotime; AT819) monoclonal antibody were diluted at 1: 2,000; and HRP-conjugated anti-rabbit IgG (Abbkine; A25022) or anti-mouse IgG (Abbkine; A25012) at 1: 5,000. The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBrightTM ECL (Advansta). The digital imaging was performed with a cold CCD camera.
3
Immunoprecipitation and Western Blot Analysis
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GC cells were lysed in ice‐cold M-PER buffer supplemented with protease and phosphatase inhibitor cocktails. After preclearing with 20 μl of protein A/G plus-agarose (sc-2003; Santa Cruz Biotechnology, USA) at 4 °C for 30 min, the cell lysates were incubated with anti‐HSPH1, anti‐SLC7A11, or IgG at 4 °C overnight on a gyratory shaker. Following incubation with 20 μl of protein A/G plus-agarose at 4 °C for 6 h, immunoprecipitated beads were washed with ice‐cold lysis buffer three times and boiled in 50 μL of 1 × SDS sample buffer for 8 min at 95 °C. Then, the immunoprecipitated protein complexes were analyzed by western blotting. To avoid the influence of the heavy chain, a specific secondary antibody against the mouse anti-rabbit IgG light chain (A25022, Abbkine Scientific, Wuhan, China) was used.
4
Western Blot Analysis of Key Proteins
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RIPA buffer (P0013B; Beyotime) was used to lyse cell and tissue samples on ice, after which a BCA kit (P0012S; Beyotime) was used to measure protein concentrations in isolated samples. Proteins were then separated by SDS‐PAGE and transferred to PVDF membranes (A10600023; GE Healthcare Life Science). Blots were blocked using 5% nonfat milk, stained overnight with Abs specific for MAP2K4 (A14781, 1:1000; ABclonal), METTL3 (96391s, 1:1000; Cell Signaling Technology), or GAPDH (2118, 1:1000; Cell Signaling Technology) at 4°C, and probed with a secondary HRP‐conjugated Ab (A25022, 1:8000; Abbkine). Protein bands were then visualized with a ChemiDoc Touch Imaging System (Bio‐Rad) and radiographic film.
5
Immunolabeling Protocol for Cartilage Markers
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The following antibodies were used in this study: rabbit anti-Col X [1:100 for immunohistochemistry (IHC); Abcam, USA; ab58632], rabbit anti-MMP-13 (1:100 for IHC; Abcam, USA; ab39012), rabbit anti-RUNX2 (1:100 for IHC; Abclone, Australia; A2851), rabbit anti-mannose receptor (1:100 for IF; Acam, USA; ab64693), rabbit anti-iNOS (1:100 for IF; Abclone, Australia; A3200), anti-rabbit IgG light chain (1:100 for IHC; Abbkine, USA; A25022), HRP-labeled goat anti-rabbit IgG H&L (1:1000 for western blots, 1:100 for IHC; Jackson Immuno Research, USA; 111-035-003), HRP-labeled goat anti-mouse IgG H&L (1:3000 for western blots; Jackson Immuno Research; 115–035-003), Alexa Fluor 594-labeled goat anti-mouse IgG H&L [1:500 for immunofluorescent labeling (I); Abcam; ab150120], and Alexa Fluor 488-labeled goat anti-rabbit IgG H&L (1:500 for IF; Abcam; ab150077).
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