United Kingdom (UK)
Recombinant monoclonal antibody to EE-tag. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the synthetic construct 3D5/EE_48.K.
Alternative Name(s) of Target: EE epitope tag (EYMPME); Glu-Glu epitope tag
Immunogen: This scFv antibody was generated by previously crystallized hexa-histidine-specific 3D5 scFv antibody. The 3D5 scFv was modified in the complementary determining region by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity.
Specificity: This antibody recognizes and binds a six amino acid long sequence (EYMPME).
Application Notes: This antibody successfully identifies protein with either internal or C-terminal EE peptides in western blot assays. When expressed in soluble form they recognize the EE peptides in ELISA (PMID:21217145; PMID:24615866).
Antibody first published in:
Pai et al. Conversion of scFv peptide-binding specificity for crystal chaperone development. Protein Eng Des Sel. (2011); 24(5): 419–428. PMID:21217145
Note on publication:
Describes the generation of this antibody.
ELISA of anti-EE-tag antibody on Multi-Tag Control Protein. Binding curves of the mouse IgG1 chimeric version of the anti-EE-tag antibody 3D5/EE_48.K (Ab02005-1.1; blue line) and isotype control (Ab00102-1.1, anti-fluorescein; green line) to an ELISA plate coated with Pr00106 (Multi-Tag Control Protein) at a concentration of 2.5 µg/ml. A 3-fold serial dilution from 1000 ng/ml was tested in the experiment. For signal detection, a 1:4000 dilution of an anti-mouse IgG1 antibody was used. Ab02005-1.1 bound to Pr00116 well.
Immunofluorescence staining of HEK293 cells with Anti-EE-tag (Ab02005) 3D5/EE_48.K Immunofluorescence analysis of paraformaldehyde fixed HEK293 transfected with EE-tag expressing plasmid (A) and non-transfected HEK293 cells (B), cells permeabilized with 0.15% Triton stained with the chimeric mouse IgG1 version of 3D5/EE_48.K (Ab02005-1.1) (1:200 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane and cytoplasmic staining. The nuclear stain is DAPI (blue).
Western Blot using Anti-EE-tag antibody 3D5/EE_48.K (Ab02005). EE-tag expressing plasmid transfected (A) and non-transfected (B) HEK293 cells lysate (3µg protein in RIPA buffer) were resolved on a SDS PAGE gel and blots were probed with the chimeric mouse version of 3D5/EE_48.K (Ab02005-1.1) at 0.03µg/ml before detection using an anti-mouse secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence.
Immunofluorescence staining of HEK293 cells with Anti-EE-tag (Ab02005) 3D5/EE_48.K Immunofluorescence analysis of paraformaldehyde fixed HEK293 transfected with EE-tag expressing plasmid (A) and non-transfected HEK293 cells (B), cells permeabilized with 0.15% Triton stained with the chimeric mouse IgG1 version of 3D5/EE_48.K (Ab02005-1.1) (1:200 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane and cytoplasmic staining. The nuclear stain is DAPI (blue).