Western blot analysis of 6x-His Epitope Tag was performed by loading various amounts of E. coli lysate, containing a multi-epitope tagged protein, per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an HRP-conjugated 6x-His Epitope Tag monoclonal antibody (Cat. No. MA1-21315-HRP) at a dilution of 1:1000 overnight at 4oC on a rocking platform and washed in TBS-0.1% Tween™ 20. Chemiluminescent detection was performed using SuperSignal West Pico (Cat. No. 34080).
Flow cytometry analysis of Histidine tag (6x-His) was done on HEK-293 cells transiently overexpressing His-IGFBP6. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with Histidine (6x-His) Mouse Monoclonal Antibody (Cat. No. 372900, red histogram) or with mouse isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor 488 Rabbit Anti-Mouse Secondary Antibody (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.
Immunofluorescence analysis of Histidine tag (6x-His) was done on HEK-293 cells. Left: Untransfected control; Right: anti-His (in red) on His-tagged fusion proteins in HEK293 cells. The cells were labeled with 6x-His Epitope Tag Monoclonal Antibody (Cat. No. MA1-21315). Both counterstained with DAPI (in blue.)
Cat. No. MA1-21315 was used in western blot to establish a process for producing HIV-1 capsid protein p24 in E.coli. Teow SY, Mualif SA, Omar TC et al. (2013) Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21 (DE3) Escherichia coli. BMC Biotechnol 13:107.
Cat. No. MA1-21315 was used in western blot to study the ability of Escherchia coli YmdB to regulate biofilm formation independently of ribonuclease III. Kim T, Lee J, Kim KS (2013) Escherichia coli YmdB regulates biofilm formation independently of its role as an RNase III modulator. BMC Microbiol 13:266.
Cat. No. MA1-21315 was used in western blot to express and purify the nucleocapsid protein of Schmallenberg virus, and raise a monclonal antibody against it. Zhang Y, Wu S, Wang J et al. (2013) Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein. Protein Expres Purif 92:1–8.
Cat. No. MA1-21315 was used in western blot to characterize SUMO cycle components in Hydra. Khan U, Mehere P, Deivasigamani S et al. (2013) The Hydra small ubiquitin-like modifier. Genesis 51:619–629.