United Kingdom (UK)
Recombinant monoclonal antibody to Softag 1. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma NT73.
UniProt Accession Number of Target Protein: P0A8T7
Alternative Name(s) of Target: rpoC; tabB; epitope tag (SLAELLNAGLGGS); DNA-directed RNA polymerase subunit beta, RNAP subunit beta, RNA polymerase subunit beta, Transcriptase subunit beta
Specificity: This antibody recognizes and binds an epitope located at the C terminus of beta subunit of the Escherichia coli RNA polymerase. It specifically binds a 13-amino-acid long sequence 'SLAELLNAGLGGS'.
Application Notes: This antibody was initially generated for purification of Escherichia coli RNA polymerase because of its ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. Such antibodies were termed as "polyol responsive" MAbs. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in the conventional purification procedure. The polyol-responsiveness of the antibody was determined by an ELISA-elution assay (PMID: 1637835). Later on the epitope of the abtibody was recognized and found to be a 13-amino-acid sequence 'SLAELLNAGLGGS' which was converted to an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag called 'Softag1' was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins (PMID: 14656522). The scFv variants of this antibody loses considerable affinity for its antigen, however it maintains similar polyol-responsiveness as the parent monoclonal antibody (PMID: 16216525).
Antibody first published in:
Thompson et al. Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase. Biochemistry (1992); 31(30):7003-8. PMID:1637835
Note on publication:
Describes the generation and characterization of the antibody.
ELISA using NT73 (Ab02006) and Multi Tag protein 2. Binding curves of the mouse IgG1 chimeric version of the Anti-Softag 1 antibody NT73 (Ab02006-1.1; purple line) and isotype control (Ab00102-1.1, anti-fluorescein; orange line) to an ELISA plate coated with Multi Tag protein 2 (Pr00565, Absolute Antibody) at a concentration of 5 ug/ml. Ab02007 antibody was titrated on a 3-fold serial dilution starting at 10,000 ng/mL. Anti-mouse IgG was used as secondary antibody for detection at a dilution of 1:4,000.
Western Blot using Anti-Softag 1 antibody NT73 (Ab02006). Softag 1 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 rabbit version of NT73 (Ab02006-23.0) at 0.03µg/ml before detection using an anti-rabbit secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence.
Immunofluorescence staining of HEK293 cells with Anti-Softag 1 (Ab02006) NT73 Immunofluorescence analysis of paraformaldehyde fixed HEK293 cells transfected with Softag 1 expressing plasmid (A) and non-transfected HEK293 cells (B), permeabilized with 0.15% Triton stained with the chimeric rabbit IgG version of NT73 (Ab02006-23.0) (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).