IDD3-Myc + LPA1-GFP, IDD13-Myc + LPA1-GFP, IDD10-Myc + LPA1-GFP, IDD3-HA + IDD13-Myc, or IDD3-HA + IDD13-Myc + LPA1-GFP were coexpressed in N. benthamiana leaves, respectively. After 36 h of expression, the protein was extracted, and Co-IP assays were performed as described previously (Kim et al. 2009b (link)). Twenty micrograms of protein from each sample were separated on a 10% SDS-PAGE gel and electrotransferred onto Immobilon-P Transfer Membranes (MILLIPORE JAPAN, Tokyo, Japan). For the subsequent western blot analysis, the following primary antibodies were used: an anti-HA antibody (1:2000; Abcam, Cambridge, MA, USA), anti-GFP antibody (1:2000; Abcam), and anti-Myc antibody (1:2000; Abcam). The membranes were incubated for an additional hour with an anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA) before the signal was detected using an ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
Anti mouse or anti rabbit horseradish peroxidase hrp conjugated secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent used to detect and visualize target proteins in various immunoassays, such as Western blotting and immunohistochemistry. The secondary antibody is conjugated with the enzyme horseradish peroxidase, which catalyzes a colorimetric or chemiluminescent reaction, allowing the detection and quantification of the target protein.
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Lab products found in correlation
Ecl western blotting detection system, by GE Healthcare (3 mentions)
Anti gfp antibody, by Merck Group (2 mentions)
Modified bradford protein assay kit, by Sangon (2 mentions)
Anti myc antibody, by Merck Group (2 mentions)
Anti ha antibody, by Abcam (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
Anti myc antibody, by Abcam (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Excell sure lock sds page electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Ivis optical imaging system, by PerkinElmer (1 mentions)
Histone 3, by Cell Signaling Technology (1 mentions)
H3k9me3, by Cell Signaling Technology (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti kras, by Abcam (1 mentions)
Anti lamin a, by Abcam (1 mentions)
Ecl blotting detection reagents, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti mouse or anti rabbit horseradish peroxidase hrp conjugated secondary antibody
1
Co-Immunoprecipitation of Myc and LPA1-GFP
2
Western Blot Analysis of CIPK31 Overexpression
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Leaf tissues collected from 1‐month‐old rice wild‐type and CIPK31 OX plants were thoroughly ground in liquid nitrogen, and 4‐week‐old Nicotiana benthamiana leaves were used for transient expression. One gram of each sample was lysed with 200 μl of 2× SDS sample buffer to extract the proteins. Samples were then centrifuged at 13,000 × g and 4°C for 20 min, and the supernatant was removed to a fresh tube. The protein content of the supernatant was quantified using modified Bradford protein assay kit (Sangon Biotech). The protein (20 μg) was separated on a 10% SDS‐PAGE gel and electrotransferred onto Immobilon‐P membrane (Millipore) for subsequent western blot analysis using the following primary antibodies: anti‐GFP antibody (1:2000; Sigma) and anti‐Myc antibody (1:2000; Sigma). The membranes were subsequently incubated for 1 h with anti‐mouse or anti‐rabbit horseradish peroxidase (HRP)‐conjugated secondary antibody (1:2000; Cell Signalling Technology), and the signal was detected using an ECL Western Blotting Detection System (GE Healthcare).
3
Protein Extraction and Immunoblotting from Rice
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Tissues collected from one-month-old rice plants were thoroughly ground in liquid nitrogen, and 1 g of each sample was lysed with 200 μL of 2× SDS sample buffer to extract the proteins. Samples were then centrifuged at 12,000 rpm and 4 °C for 10 min, and the supernatant was removed to a fresh tube. The protein content of the supernatant was quantified using Modified Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China). The protein (20 µg) was separated on a 10% SDS-PAGE gel and electrotransferred onto Immobilon-P Transfer Membrane (MILLIPORE JAPAN, Tokyo, Japan) for subsequent Western blot analysis, using the following primary antibodies: anti-SLR1 antibody (1:2000; Abclonal, Wuhan, China), anti-GFP antibody (1:2000; Sigma, Tokyo, Japan), and anti-Myc antibody (1:2000; Sigma). The membranes were subsequently incubated for an hour with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA), and the signal was detected using an ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
4
Proteomic Analysis of TEVs
5
Western Blot Analysis of Protein Expression and Modifications
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Cells were collected, washed, and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing Protease and Phosphatase Inhibitor (Thermo Fisher Scientific). A bicinchoninic acid (BCA) kit (CWBio) was then used to detect the concentration of protein. Equivalent amounts of protein were separated on 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was then blocked with Tris-buffered saline with Tween 20 (TBST) buffer containing 5% skim milk powder and incubated with corresponding primary antibodies at 4 °C overnight. The primary antibodies used were anti-p65 (Cell Signaling Technology), anti-IκBα (Cell Signaling Technology), anti-p50 (Cell Signaling Technology), anti-Histone-H3 (Abcam), anti-β-actin (Abcam), anti-Flag (Cell Signaling Technology), GAPDH (Cell Signaling Technology), anti-L-Lactyl Lysine (PTM Bio Inc), anti-Lactyl-Histone H3 (Lys18) Rabbit mAb (PTM Bio Inc), anti-KRAS (Abcam), anti-KRASG12D (Abcam), anti-Lamin A (Abcam). Membranes were then washed with TBST three times and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) for 1 h at room temperature. Signals were developed with ECL Blotting Detection Reagents (Thermo Fisher Scientific).
6
Immunoblotting of Cell Signaling Proteins
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