Western blot analysis of recombinant N-terminal His-tagged turbo GFP was performed by loading the indicated amounts of protein, and 15ul of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with StartingBlock (PBS) Blocking Buffer (Product # 37538) for at least 1 hour at room temperature. N-terminal His-tagged turbo GFP was detected at ~37kD using a 6x-His Epitope Tag monoclonal antibody (Product # MA1-135) at a concentration of 2ug/ml in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody (Product # 31439) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
|Tested species reactivity||Tag|
|Host / Isotype||Mouse / IgG|
|Immunogen||Mixture of N-terminal and C-terminal 6x-His peptides, CG-HHHHHH and HHHHHH-GC|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|ELISA (ELISA)||25-500 ng/ml|
|Immunoprecipitation (IP)||2 ug|
|Western Blot (WB)||1:250-1:1000|
* 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.
By Western blot, MA1-135 detects both N-terminal and C-terminal His-tagged proteins.
Epitope tagging is a technique in which a known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which an antibody is available, the technique makes it possible to detect proteins for which no antibody is available. HIS epitope tagged proteins that contain a stretch of histidine residues at the carboxyl or amino terminus is used for purification of recombinant proteins by means of metal chelate chromatography due to metal binding properties of histidine clusters on His tagged proteins.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.