Journal: Biochemical and biophysical research communications

Article Title: Characterization of an anti-FLAG antibody binding protein in V. cholerae

doi: 10.1016/j.bbrc.2020.05.169

Figure Lengend Snippet: (A) Construction of 3×FLAG tag fusion proteins. 3×FLAG peptide was fused after N-terminus signal sequences of Protein A and B, or at C-terminus region of Protein C for quantification of protein levels. (B) Detection of non-specific protein by anti-FLAG antibody in both wild type and 3×FLAG fusion strains (C) Growth phase dependent induction of anti-FLAG antibody binding protein. Lag, lag phase; Log, exponential phase; Sta, stationary phase.

Article Snippet: Wild type V. cholerae and 3×FLAG tag+ ProteinA fusion strains were grown in LB medium to mid-logarithmic phase (at an OD 600 ~0.5) and then 20 ml cell cultures were spun down at 4830 rcf for 10 min.

Techniques: Binding Assay

Journal: Biochemical and biophysical research communications

Article Title: Characterization of an anti-FLAG antibody binding protein in V. cholerae

doi: 10.1016/j.bbrc.2020.05.169

Figure Lengend Snippet: (A-B) Silver-stained (A) or zinc-stained (B) protein gel demonstrating effective IP with FLAG antibody resin. (C) Western Blot showing excision of non-specific protein-containing region from the gel in (B). Each half volume of eluted sample from IP was loaded in two wells of 10% SDS-PAGE gel; one for positive control of western blotting and the other for in-gel tryptic digestion. (D) Venn diagram showing number of detected total proteins from IP/LC-MS/MS of the size-selected gel band in wild type and 3×FLAG fusion strain.

Article Snippet: Wild type V. cholerae and 3×FLAG tag+ ProteinA fusion strains were grown in LB medium to mid-logarithmic phase (at an OD 600 ~0.5) and then 20 ml cell cultures were spun down at 4830 rcf for 10 min.

Techniques: Staining, Western Blot, SDS Page, Positive Control, Liquid Chromatography with Mass Spectroscopy

Journal: Biochemical and biophysical research communications

Article Title: Characterization of an anti-FLAG antibody binding protein in V. cholerae

doi: 10.1016/j.bbrc.2020.05.169

Figure Lengend Snippet: (A) Construction of overexpression vectors for 6xHis tag fusion proteins. A 6xHis tag was fused to the N-terminus region of all proteins (OmpA, Porin4, VxrB, ShyA, and Bacillus subtilis MntR) and overproduced in E. coli followed by detection via anti-His and anti-Flag Western Blot. Vec, empty vector. Black stars or red stars indicate bands recognized by anti-His antibody or anti-FLAG antibody, respectively. Blue star presents the band detected by both antibodies. (B) Amino acid sequence alignment between 3×FLAG tag and the relevant parts of Porin4 protein reveals the putatively FLAG-antibody-reactive Porin4 tag. Identical residues are highlighted yellow.

Article Snippet: Wild type V. cholerae and 3×FLAG tag+ ProteinA fusion strains were grown in LB medium to mid-logarithmic phase (at an OD 600 ~0.5) and then 20 ml cell cultures were spun down at 4830 rcf for 10 min.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation

doi: 10.1007/s00018-022-04663-x

Figure Lengend Snippet: Naturally occurring nitrated CXCL8 in BAL samples from suspected-VAP patients. a Structure of HuCAL antibody, bivalent monoclonal antibody consisting of the antigen-binding fragment with two antigen recognition sites connected by an alkaline phosphatase dimerization domain with FLAG ® and Histidine 6 tags for detection. b Specificity of the antibody was tested via dot blot with wild type or nitrated CXCL8 detected with either AHC0881, which recognizes both variants of CXCL8 and HuCAL AbD31649.1, which recognizes only the nitrated variant and c ELISA. Nitrated BSA was used as a control to test cross-reactivity with a non-specific nitrated protein at the highest concentration. Detection was carried out using a biotinylated anti-histidine 6 secondary antibody (MCA1396B, Bio-Rad), streptavidin-HRP and TMB substrate, and the plate was read at 450 nm N = 3. d Identification of CXCL8, nitrated proteins and naturally occurring nitrated-CXCL8 using western blot analysis on BAL samples. d – f Lanes 1 and 2: healthy controls. Lanes 3–7: BAL samples from VAP patients. Gels were run in triplicate and the resulting membranes after protein transfer were probed with, d rabbit polyclonal anti-CXCL8 antibody (AHC0881), e mouse monoclonal anti-3-nitrotyrosine antibody (HM5001) and f HuCAL ® antibody (AbD31649.1). g Immunoprecipitation of BAL samples using 3-NT affinity columns and subsequently detecting for the presence of CXCL8. Lane 1: CXCL8, Lane 2: nitrated-CXCL8. Lane 3: blank. Lane 4: pulldown. Lane 5: blank. Lane 6: unbound fractions. Lane 7: blank

Article Snippet: Potential candidate antibodies that display > fivefold higher specificity for nitrated over wild type CXCL8 were produced as bivalent Fab-bacterial alkaline phosphatase fusion antibodies with FLAG ® and Histidine 6 tag.

Techniques: Binding Assay, Dot Blot, Variant Assay, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Western Blot, Immunoprecipitation