GFP control protein was synthesized with an amino terminal 6XHIS tag and was expressed in E. coli and purified by sequential hydrophobic interaction chromatography, DEAE chromatography and size-exclusion chromatography. Assay by immunoelectrophoresis resulted in a single precipitin arc against Anti-GFP. Analysis by SDS-PAGE resulted in a pattern consistent with purified GFP and was estimated to be greater than 90% pure.
Formulierung:
Liquid (sterile filtered)
Application Verdünnung:
ELISA: User Optimized, IP: User Optimized, WB: User Optimized
Anwendungsbeschreibung:
Application Notes: GFP Control is suitable as a control for polyclonal or monoclonal Anti-GFP in immunological assays. Researchers should determine optimal titers for applications. The molecular weight of GFP is 28 kDa. Green Fluorescent Protein has been tested in SDS-page, western blot, and immunoprecipitation
Immunoprecipitation/Western Blot using GFP Protein. Lane 1: Opal Prestained Molecular Weight Marker. Lane 2: GFP Input (p/n orb345957) Reduced [10 µl]. Primary IP Antibody: Mouse Anti-GFP (p/n orb345329) at 10 µg overnight at 2-8C. Secondary Antibody: TrueBlot Anti-Mouse Ig IP Agarose Beads at 500 µg for 1hr at RT. Buffer: BlockOut Buffer (p/n orb348644) for 30 mins at RT. Exposure: 7 sec.
SDS-PAGE results of Recombinant GFP Control Protein. Lane 1: Recombinant GFP Control Protein Reduced (1.0 µg). Lane 2: Opal Prestained Molecular Weight Marker. Lane 3: Recombinant GFP Control Protein Non-Reduced (1.0 µg). Predicted MW: ~28kDa. Observed MW: ~28kDa doublet, ~60kDa dimer. 4-20% Gel Coomassie Stained.
Western Blot of anti-GFP monoclonal antibody. Lane 1: 64pg of recombinant GFP protein (p/n orb345957) were spiked into a HeLa cell-derived lysates (p/n orb348668). Lane 2: 32pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 3: 16pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 4: 8pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 5: 4pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 6: 2pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 7: 1g of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Lane 8: 0pg of recombinant GFP protein were spiked into a HeLa cell-derived lysates. Primary antibody: anti-GFP monoclonal antibody at 1:400 for overnight at 4C. Secondary antibody: HRP-conjugated anti-Mouse IgG (p/n orb347544) was performed at a dilution of 1:20000 for 1h at 4C. Block: TTBS (p/n orb348540) supplemented with 1% BSA for 1 h at 4C. Predicted/Observed size: 27 kDa for GFP. Other band(s): none.
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