Western 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 10ul 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 (0.8mg/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).
|Tested species reactivity||Tag|
|Host / Isotype||Rat / IgG2a|
|Immunogen||Recombinant fusion proteins of mouse Langerin/CD207|
|Tested Applications||Dilution *|
|Western Blot (WB)||1ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
The MA1-142 antibody (clone L5) has been successfully used as a detection antibody in a direct ELISA with FLAG-tagged protein-coated plates.
similar epitope as the FLAG tag from Sigma
The MA1-142 immunogen is recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. (J Immunol Methods. 2008 Feb 29;331(1-2):27-38. Epub 2007 Nov 26.)
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. The FLAG tag's structure has been optimized for compatibility with the proteins it is attached to, in that it is more hydrophilic than other common epitope tags and therefore less likely to denature or inactivate proteins to which it is appended. A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a FLAG-tag to this protein allows one to follow the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis.
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