| DYKDDDDK Tag Antibody (MA1-142) in ELISADirect ELISA analysis of a FLAG Tag monoclonal antibody (Product # MA1-142) was performed by loading 100 µL per well of recombinant N-terminal FLAG-tagged ubiquitin protein at a concentration of 1 µg/mL overnight at 40C on polystyrene 96-well strip plates (Product # 15031). The plate was washed 3 times with ELISA Wash Buffer (Product # N503), and 100 µL of a FLAG Tag monoclonal antibody (Product # MA1-142) was added to wells in duplicate at 500, 250, 125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.9, 0.4 and 0 ng/mL concentrations, and the samples were incubated for 2 hours at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-rat IgG antibody (Product # 31470) at a dilution of 1:10,000 for 60 minutes at room temperature. Detection was performed by adding 100 µL of 1-Step Ultra TMB ELISA substrate (Product # 34028) per well and incubating for 15 minutes at room temperature in the dark. The reaction was stopped with 100 µL per well of 0.16M sulfuric acid (Product # N600). Absorbance's were read on a spectrophotometer at 450-550 nm.DYKDDDDK Tag Antibody (MA1-142) in ELISADirect ELISA analysis of a FLAG Tag monoclonal antibody (Product # MA1-142) was performed by loading 100 µL per well of recombinant C-terminal FLAG-tagged bacterial (E. coli) alkaline phosphatase fusion protein at a concentration of 1 µg/mL overnight at 40C on polystyrene 96-well strip plates (Product # 15031). The plate was washed 3 times with ELISA Wash Buffer (Product # N503), and 100 µL of a FLAG Tag monoclonal antibody (Product # MA1-142) was added to wells in duplicate at 500, 250, 125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.9, 0.4 and 0 ng/mL concentrations, and the samples were incubated for 2 hours at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-rat IgG antibody (Product # 31470) at a dilution of 1:10,000 for 60 minutes at room temperature. Detection was performed by adding 100 µL of 1-Step Ultra TMB ELISA substrate (Product # 34028) per well and incubating for 15 minutes at room temperature in the dark. The reaction was stopped with 100 µL per well of 0.16M sulfuric acid (Product # N600). Absorbance's were read on a spectrophotometer at 450-550 nm.DYKDDDDK Tag Antibody (MA1-142) in WBWestern blot analysis of a FLAG Tag monoclonal antibody was performed by loading 50, 25, 12.5, 6.25, 3.25, and 1.5 ng of recombinant N-terminal FLAG-tagged ubiquitin protein and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for at least 1 hour. The membrane was probed with a FLAG Tag monoclonal antibody (Product # MA1-142) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a pre-diluted (0.8 mg/mL) HRP-conjugated goat anti-rat IgG secondary antibody (Product # 31470) at a dilution of 1:10000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). DYKDDDDK Tag Antibody (MA1-142) in WBWestern blot analysis of a FLAG Tag monoclonal antibody was performed by loading 50, 25, 12.5, 6.25, 3.25 and 1.5 ng of recombinant C-terminal FLAG-tagged bacterial (E. coli) alkaline phosphatase fusion protein and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for at least 1 hour. The membrane was probed with a FLAG Tag monoclonal antibody (Product # MA1-142) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a pre-diluted (0.8 mg/mL) HRP-conjugated goat anti-rat IgG secondary antibody (Product # 31470) at a dilution of 1:10000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). |
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