PEPC | phosphoenolpyruvate carboxylase positive control/quantitation standard
AS09 458S | protein standard for quantitation and positive control
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Standard curve: 3 loads are recommended (0.5, 2 and 4μl).
Positive control: a 2 μl load per well is optimal for most chemiluminescent detection systems. Higher standard concentration needs to be used to allow detection by Coomasie stains. Such gels with higher standard concentration can not be used for quantitation using chemiluminescence.
|Expected | apparent MW||
110 | 105 kDa
|Not reactive in|
Concentration: after re-constitution with sterile milliQ water final concentration of the standard is 0.15 pmoles/µl
Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50 mM DTT.
|Selected references||to be added when available, product released in October 2014.|
5 ug total protein from 10-day old Zea mays leaves was loaded into Bolt 4-12% Bis-Tris Plus polyacrylamide gel (Invitrogen) in NuPAGE LDS sample buffer (1X, Invitrogen) and dithiothreitol (50 mM) along with four concentrations of PEPC standard (100, 200, 400 and 800 fmoles). The same concentrations of standard from lyophilized sample were also loaded into the same gel to evaluate the effectiveness of lyophilization process. Proteins were separated in Bolt MOPS SDS running buffer (1X, Invitrogen) at 200 V for 32 min using a Bolt Mini Gel Tank (Invitrogen). Proteins were then transferred onto 0.2-μm polyvinylidene fluoride membranes (PVDF, Immobilon) for 80 min at 20 V in NuPAGE transfer buffer (1X, Invitrogen) containing methanol (10%, v/v) and Bolt Antioxidant (Invitrogen) using Bolt Mini Blot Module (Invitrogen). Following the transfer, membrane was blocked for 1 h in 2% (w/v) membrane blocking agent (GE Healthcare) dissolved in Tris-buffered saline solution containing Tween-20 (TBS-T; Tris, 20 mM; NaCl, 137 mM; Tween-20, 0.1% v/v). The membrane was treated with PEPC antibody (1:10,000, w/v in blocking solution, AS09 458) for 1h. Membrane was washed with TBS-T twice briefly, then once for 15 min and three times for 5 min each. Membrane was then incubated for 1 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Agrisera; 1:25,000, w/v in blocking solution, AS09 602) followed by washing as described above. The signals were detected using ECL reagent and visualized using the Molecular Imager VersaDoc MP 4000 System (Bio-Rad).
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