A partial ARFTF17 protein fragment from the 480th to 644th amino acid was used by ABclonal (Wuhan, China) to make antibodies. To analyze accumulation of ARFTF17 protein in B73 and different fka1-1 mutants, total protein was extracted from the pericarp and endosperm at eight and 12 DAP with the non-zein buffer52 (link). Twenty μg of total protein was separated by 10% SDS-PAGE and transferred electrophoretically to a PVDF membrane. Immunodetection used ARFTF17 antibodies at a concentration of 1:1,000 at 4 °C overnight, followed by a secondary anti-rabbit-HRP at a concentration of 1:5,000 (Abmart, catalog number: M21002L). To detect the control protein, ACTIN, a primary antibody, mouse monoclonal ACTIN antibody (Abmart, catalog number: M20009L) and a secondary antibody, anti-mouse IgG-HRP (Abmart, catalog number M21001L), were used. To examine FLAG in fka1-1; ARFTF17pro:Flag-ARFTF17 plants, total protein was extracted from the pericarp. Immunoblotting used anti-FLAG (Sigma, A8592) as the primary antibody at a dilution of 1:1,000, and anti-mouse IgG-HRP (Abmart, M21001L) as the secondary antibody at a dilution of 1:5,000. The membranes were treated with a chemiluminescence substrate (Invitrogen, catalog number: WP20005), and the immunoreactive bands were detected using Tanon-5200 system.
Anti mouse igg hrp
Manufactured by Abmart
Sourced in United States
Anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassay applications.
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Lab products found in correlation
Mouse monoclonal actin antibody, by Abmart (1 mentions)
Anti flag, by Merck Group (1 mentions)
Western blotting detection reagent, by GE Healthcare (1 mentions)
Mouse anti β tubulin, by Abmart (1 mentions)
Rabbit anti ucp1, by Alpha Diagnostic (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
2 protocols using anti mouse igg hrp
1
Antibody-Based Immunodetection of ARFTF17
2
Western Blotting of UCP1 Expression
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Differentiated cells were washed with PBS, lysed, and boiled at 95–100 °C for 5 min in sample buffer (50 mM Tris-HCl, 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenyl blue (pH 6.8)). The protein lysates were separated through SDS-PAGE before being transferred to PVDF membranes. The membranes were first blocked in TBST (TBS with 0.1% Tween 20) containing 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C in TBST buffer containing mouse anti-β-tubulin (1:6000; Abmart, China) or rabbit anti-UCP1 (1:500; Alpha Diagnostic International, USA). The membranes were washed three times with TBST and incubated with anti-rabbit IgG HRP (1:8000; Cell Signaling, USA) or anti-mouse IgG HRP (1:8000; Abmart, China) for 1 h at room temperature. After washing three times with TBST, immunostaining was visualized using Western blotting detection reagents (GE Healthcare, USA).
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