Two-week-old seedlings were treated with 1 μM solutions of AtPep1 and then harvested in liquid nitrogen after 0, 5, 10, 30 and 60 min. Proteins were extracted with Lacus buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 15 mM EGTA, 100 mM NaCl, 1 mM sodium fluoride, 1 mM sodium molybdate, 0.5 mM Na3VO4, 30 mM β-glycerol-phosphate, 0.1% Triton-X 100), as described previously70 (link). The homogenized protein samples were centrifuged at 21.000 × g for 20 min at 4°C. 5x SDS loading buffer was added into each supernatant and 12 μl were subjected to immunoblot analysis. After transfer, protein was blocked for MAPK activation detection with 5% BSA for 1 hour. After washing three times in TBS-T, the membrane was incubated overnight at 4°C in TBS-T and α-p44/42 MAPK (Erk1/2) antibody (1:5000, CST). Following three washes with TBS-T, the membrane was incubated in α-rabbit-HRP (1:5000, Sigma) for 2 hours. Phosphorylated MAP kinases 3, 4/11, and 6 were detected using Pierce ECL western blotting substrate (Thermo Fisher).
α rabbit hrp
Manufactured by Merck Group
α-Rabbit HRP is a laboratory reagent used in various immunoassay techniques. It contains rabbit-derived horseradish peroxidase (HRP) conjugate, which enables detection and quantification of target analytes in biological samples.
Automatically generated - may contain errors
4 protocols using α rabbit hrp
1
AtPep1 Induces MAPK Activation
2
Western Blot Analysis of Protein Expression
3
Western Blot Analysis of Protein Expression
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20 μg of protein was loaded for SDS PAGE and transferred onto a nitrocellulose membrane for western blots. Primary antibodies (α-FLAG; Sigma-Aldrich cat. #F7425 and α-GAPDH; Cell Signaling Technology cat. #14C10) were used at a 1:1000 dilution in TBST + 5% Milk. Secondary α-Rabbit HRP (Sigma-Aldrich cat. #A6154) was used at a 1:5000 dilution in TBST + 5% Milk. Membranes were exposed after addition of ECL (Bio-Rad cat. #170-5060).
4
Expression and Localization of Recombinant GAPDH
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Electroporation in M. smegmatis and M.tb H37Ra was done as described previously [9 (link)]. A single colony from each transformation was cultured in 7H9 media supplemented with OADC and 50 µg/ml hygromycin up to log phase. Sub cellular fractions were prepared and analysed for the presence of recombinant GAPDH by western blotting [9 (link)]. Briefly, 40 µg each of cytosol, cell membrane and cell wall fractions from M. smegmatis and M.tb H37Ra transformed with either plasmid were denatured by heating for 10 min at 95 °C and loaded on a 10 % SDS-PAGE. Control fractions of untransformed M. smegmatis and M.tb H37Ra were run alongside. Resolved proteins were either stained with coomassie blue or were transferred onto nitrocellulose membrane. Blots were probed with mouse monoclonal α-His (Sigma) for 1 h followed by incubation with goat α-mouse IgG-HRP (1:10,000 for 1 h), washed and developed with TMB/H2O2. Blots were also stripped and re-probed with 1:1000 dilution of rabbit polyclonal α-GAPDH followed by detection with 1:16,000 dilution of α-Rabbit-HRP (Sigma).
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