Fifty adult hermaphrodites from strain AF16 [wild-type C. briggsae], strain TY5774 Cbr dpy-27(y706), 3xFLAG-tagged Cbr dpy-27, or strain TY5005 [Cbr dpy-27(y436)] were picked into 25 µL of water, diluted with 25 µL of 2 x SDS Sample Buffer, and heat denatured at 98 °C for 4 min. Samples (20 µL) were fractionated with 3–8% Tris Acetate SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes using standard conditions (60 min at 100 V). Membranes were immunoblotted with either rabbit polyclonal anti-DPY-27 (this study) or rabbit polyclonal anti-MIX antibody (this study). Following incubation with a primary antibody, membranes were incubated with a secondary donkey anti-rabbit HRP antibody (Jackson ImmunoResearch, #711-035-152, RRID: AB_10015282 ). Nitrocellulose membranes were then incubated in WesternBright Sirius ECL solution (Advansta Corporation, #K-12043-D20) for 2 min, and the chemiluminescence signal was acquired using Image Lab software (Bio-Rad).
Westernbright sirius ecl solution
Manufactured by Advansta
Sourced in United States
WesternBright Sirius ECL solution is a chemiluminescent substrate used for the detection of protein bands in Western blot analysis. It is designed to provide a sensitive and reliable method for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Horseradish peroxidase hrp conjugated anti mouse igg, by Cell Signaling Technology (2 mentions)
Donkey anti rabbit hrp antibody, by Jackson ImmunoResearch (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
4 protocols using westernbright sirius ecl solution
1
Immunoblotting of C. briggsae DPY-27 and MIX
2
Western Blot Analysis of Myc-Tagged Proteins
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Total protein was isolated from –100 mg of seedlings using a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1 mM DTT and 1×cOmplete EDTA-free Protease Inhibitor Cocktail (Roche). Fifty micrograms of total protein were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were transferred to PVDF membranes (Amersham Biosciences) and probed with an anti-myc (MBL; #M192-3; 1:10,000 dilution) antibody overnight at 4 °C. The samples were then probed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling, Danvers, MA, USA; #7076; 1:10,000 dilution) antibody at room temperature. The signals were detected with ImageQuant LAS 4000 (GE Healthcare) using WesternBright Sirius ECL solution (Advansta, San Jose, CA, USA).
3
Detecting Myc-tagged Proteins in Plant Seedlings
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Total protein was isolated from ~100 mg of seedlings using buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1 mM DTT and the 1X cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche). 50 μg of total protein was loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were transferred to PVDF membranes (Amersham Biosciences) and probed with anti-myc (MBL, #M192-3; 1:10000 dilution) antibody overnight at 4°C. Then, the samples were probed with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling, #7076; 1:10000 dilution) antibody at room temperature. The signals were detected by ImageQuant™ LAS 4000 (GE Healthcare) using WesternBright™ Sirius ECL solution (Advansta).
4
CCA1 and LHY Protein Analysis
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Vernalized or non-vernalized pCCA1:CCA1-HA-YFP and pLHY:LHY-FLAG seedlings were harvested at each time point. Whole seedlings for each transgenic line were collected for total protein extraction. Total proteins were prepared from 100 mg of harvested samples in protein extraction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 , 1 mM ethylenediaminetetraacetic acid (EDTA), 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, 1× cOmplete Mini, and EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). Total proteins (50 µg) were separated by sodium dodecyl sulfate (SDS)-PAGE. The proteins were transferred to PVDF membranes (Amersham Biosciences, Amersham, UK) and probed with anti-HA (Cell Signaling, Danvers, MA, USA; #3724; 1:5,000 dilution) or anti-FLAG (Sigma-Aldrich, St Louis, MO, USA; #F3165; 1:10,000 dilution) antibodies overnight at 4°C. The samples were then probed with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling; #7074; 1:10,000 dilution) or anti-mouse IgG (Cell Signaling; #7076; used at 1:10,000 dilution) antibodies at room temperature. The signals were detected using ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA) with WesternBright™ Sirius ECL solution (Advansta San Jose, CA, USA).
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