Total proteins were extracted from seedlings using 1× Laemmli sample buffer (Bio-Rad, 1610737) and immunoblotting was carried out with an HRP-conjugated anti-GFP antibody (Miltenyi Biotec, 130-091-83, 1:3000 dilution) or a Rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956, 1:1000 dilution) followed by anti-rabbit IgG-HRP (Cell Signaling Technology, 7074, 1:3000 dilution). Either cross-reacting bands or proteins stained by Ponceaus S (Sigma-Aldrich, P3504; 0.1% w/v in 5% acetic acid) were used as loading controls. Chemiluminescent substrate (Thermo Fisher Scientific, 34075) was used for signal detection.
Hrp conjugated anti gfp antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
The HRP-conjugated anti-GFP antibody is a secondary antibody that specifically binds to green fluorescent protein (GFP). It is conjugated with horseradish peroxidase (HRP), which allows for colorimetric or chemiluminescent detection of GFP-tagged proteins in various applications such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Rabbit monoclonal anti gfp antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Amersham ecl select western blotting detection reagent, by GE Healthcare (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
3 protocols using hrp conjugated anti gfp antibody
1
Extracting and Analyzing Seedling Proteins
2
GFP Western Blot Protocol
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Leaf disks (60 mg) were collected, frozen in liquid nitrogen and ground to a fine powder. Proteins were extracted in 60 μL of extraction buffer [0.35 M Tris‐HCl (pH 6.8), 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue]. Total protein (10 μL) was separated by 10% SDS‐PAGE. Separated proteins were transferred onto a membrane and incubated with an HRP‐conjugated anti‐GFP antibody (1:5000; Miltenyi Biotec, Bergisch Gladbach, Germany). Immunodetection was performed using an ECL Prime western blotting detection reagent (GE Healthcare, Marlborough, MA, USA).
3
Immunoblot Analysis of GFP, BES1, and Actin
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Immunoblot analysis was done as described previously (Albertos et al, 2015 (link)) using HRP‐conjugated anti‐GFP antibody (Miltenyi Biotec), rabbit anti‐BES1 (Yin et al, 2002 (link)), and mouse anti‐Actin (Sigma) as primary antibodies and alkaline phosphatase‐conjugated goat anti‐rabbit or anti‐mouse IgG (Sigma‐Aldrich) as secondary antibodies. Detection was performed with enhanced chemiluminescence using the Amersham ECL Select Western Blotting Detection reagent (GE Healthcare) or PhosphaGLO Reserve AP Substrat (Medac Diagnostic). For loading control, the membranes were stained with the Coomassie Brilliant Blue dye.
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