For western blotting, 20 μg of cellular protein extracts were used [23 (link)]. We used rabbit anti-Akt antibody, anti-S473- and anti-T308-phospho-specific Akt antibodies, anti-PDK1 antibody, anti-S241-phospho-specific PDK1 antibody, anti-JNK antibody, anti-T183/Y185-phospho-specific JNK antibody, anti-p38 antibody, anti-phospho-specific p38 antibody (Cell Signaling Technology, USA), and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA). Relative protein amounts were evaluated using Chemidoc XRS system (Bio-Rad, USA) and ImageJ tool (National Institute of Health, USA).
Horseradish peroxidase conjugated anti rabbit igg secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody is a laboratory reagent used to detect the presence of rabbit immunoglobulin G (IgG) in various experimental techniques. The horseradish peroxidase enzyme is covalently attached to the anti-rabbit IgG antibody, allowing for the visualization of the target protein through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
C jun, by Cell Signaling Technology (3 mentions)
Anti m6a antibody, by Synaptic Systems (3 mentions)
Hybond n membrane, by Cytiva (3 mentions)
Anti total ampk, by Cell Signaling Technology (2 mentions)
Anti phosphorylated ampk, by Cell Signaling Technology (2 mentions)
Ecl reagent, by GE Healthcare (2 mentions)
Anti beta actin, by AbFrontier (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Chemiluminescent film, by Thomas Scientific (2 mentions)
Pser73 jun, by Cell Signaling Technology (2 mentions)
Amersham hybond n membrane, by GE Healthcare (2 mentions)
Bio dot apparatus, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
24 protocols using horseradish peroxidase conjugated anti rabbit igg secondary antibody
1
Western Blotting Analysis of Cellular Signaling
2
Western Blotting Protocol for AMPK Quantification
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The methods used for Western blotting have been described previously [2 (link),22 (link)]. Proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane (Merck KGaA) via a wet transfer system. The membranes were then maintained overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (AbFrontier, Seoul, South Korea). The membranes were then maintained with an anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Antibody-antigen complexes were visualized using the EzWestLumi Plus Detection Kit (ATTO Corporation, Tokyo, Japan). Luminescent images were obtained using a LuminoGraph II Imaging System (ATTO Corporation).
3
Immunoblot Analysis of DNMT3A and Tubulin
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The protocol was used as previously described [4 (link)]. All primary antibodies were incubated with the membrane at 4°C overnight: DNMT3A (Santa Cruz Biotechnology, sc-20703), and tubulin (Sigma, T6199). Membranes were washed with 1×TBST and incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology).
4
AMPK Activation Assay Using Western Blot
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Proteins were extracted using a buffer containing 25 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, and protease inhibitor cocktail (Roche Korea, Seoul, Korea). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK, and anti-beta-actin (AbFrontier, Seoul, Korea). Membranes were then probed with the anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Antibody–antigen complexes were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Korea). For the quantification of phospho-AMPK and total-AMPK, ATTO image analysis software (CS analyzer 4, Tokyo, Japan) was used.
5
Protein Extraction and Western Blot Analysis
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Proteins were extracted using the lysis buffer containing 25 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na VO, and a protease inhibitor cocktail (Roche Korea, Seoul, Republic of Korea). Proteins were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK, anti-total Akt, anti-phosphorylated Akt (Cell Signaling Technology, Beverly, MA, USA), PTP-MEG2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (AbFrontier, Seoul, Republic of Korea). Membranes were then probed with an anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Antibody–antigen complexes were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Republic of Korea).
6
Western Blot Quantification of SIRT1 and NF-κB
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The Western blot procedure was described in detail by Mikłosz et al. [56 (link)] Briefly, tissue homogenate containing 30 µg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (0.75 A for 1 h). Then, membranes were blocked in Tris Buffer Saline Tween 20 (TBST; 20 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat dry milk (90 min at room temperature). Membranes were incubated overnight with primary antibodies: SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-nuclear factor-κB (NF-κB) (Cell Signaling Technology, Leiden, The Netherlands). Next, SIRT1 and NFκB were detected with antirabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The protein bands were visualized using a chemiluminescence substrate (Thermo Scientific, Waltham, MA, USA) and quantified by densitometry (Bio-Rad Systems). The protein expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology) expression.
7
Quantifying Protein Expression After Transfection or Irradiation
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Cell lines at various times post-transfection or after γ-irradiation were collected and lysed in radioimmunoprecipitation assay buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Roche) as previously described36 (link). 20μg protein was resolved onto 4-20% mini-PROTEAN TGX gels (Bio-Rad), and transferred to PVDF membranes. Primary antibodies were used to detect pSer73-JUN (1:1000, Cell Signaling Technology, #3270), c-JUN (1:1000, Cell Signaling Technology, #9165), and β-actin (1:1000, Cell Signaling Technology, #4970). Following incubation with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:10,000, Santa Cruz, #2030), expression was detected via Clarity Western ECL (BioRad) and chemiluminescent film (Denville Scientific). Band intensities were quantified using NIH Image J software.
8
Quantifying Protein Expression After Transfection or Irradiation
9
Western Blot Analysis of Renal Proteins
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Western blot analysis was performed as previously described (24 (link)). Renal tissues were homogenized with ice-cold lysis buffer and were centrifuged at 10,000 × g for 15 min at 4°C. Equal amounts (100 µg) of protein were loaded and separated by 10% SDS-PAGE. Separated proteins were then transferred onto nitrocellulose paper (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A sample of total protein from the lysate (20 µg) was separated and blotted. The membranes were incubated with the following primary antibodies: Anti-RAGE (1:250, cat no. MAB1179; R&D Systems, Inc., Indianapolis, IN, USA), anti-nephrin (1:1,000, cat no. ab58968; Abcam, Cambridge, UK), anti-podocin (1:1,000, cat no. sc-21009; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin (1:500, cat no. ab8226; Abcam) overnight at 4°C, and then reacted with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:5,000, cat no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 1.5 h. Signal detection was performed via exposure of the blot to enhanced diaminobenzidenecolor reagents (OriGene Technologies, Inc., Rockville, MD, USA) for 5 min. Quantification of the luminosity of each identified protein band was performed using Adobe Photoshop software (AdobePhotoshop 7.0; Adobe Systems, Inc., San Jose, CA, USA).
10
m6A Dot Blot Assay Protocol
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The m6A dot blot assay was conducted as previously described. Briefly, the indicated amount of total RNA was denatured in incubation buffer at a 1:3 ratio (65.7% formamide, 7.77% formaldehyde and 1.33 × MOPS) at 65°C for 5 min, followed by chilling on ice and mixing with 20 × SSC buffer at a 1:1 ratio. RNA samples were transferred to an Amersham Hybond-N+ membrane (GE Healthcare) with a Bio-Dot Apparatus (Bio-Rad) and auto-crosslinked 3 times with the auto-crosslinking mode. After UV crosslinking, the membrane was stained in 0.3 M sodium acetate with 0.02% methylene blue (MB). Then, the membrane was washed with 1 × PBST buffer, blocked in 5% nonfat milk in PBST for 1 h at room temperature and incubated with anti-m6A antibody (202003, 1:1,000; Synaptic Systems) overnight at 4°C. The membrane was washed according to the standard protocol. After incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology), the membrane was visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).
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