anti epo polyclonal antibody  (R&D Systems)

Journal: Journal of Sport and Health Science

Article Title: Reverse–normal immunopurification: An effective approach for purifying recombinant erythropoietin from its analogues in doping analysis

doi: 10.1016/j.jshs.2025.101062

Figure Lengend Snippet: Development of the reverse–normal immunopurification method coupled with western blotting. (A) Schematic of the reverse–normal immunopurification method. VAR-EPO is designed to be removed and analyzed separately from WT-EPO and rEPO. With this method, rEPO can be detected without interference of VAR-EPO. (B) EC50 curve of antibody. Each antigen, including the differential peptide between WT-EPO and VAR-EPO (DP), rVAR-EPO, and rEPO, was coated in a series of 2-fold dilutions ranging from 1 ng to 7.81 pg per well. Each well contained 1 µg of antibody. The anti-VAR mAb specifically recognizes DP and rVAR-EPO, with no cross-reactivity to rEPO. Anti-VAR mAb: TYJ9-R0016 (in-house developed); anti-EPO pAb: AB-286-NA (R&D systems). (C) Surface plasmon resonance (SPR) assay to determine the affinity between anti-VAR mAb (TYJ9-R0016, developed in-house) and rVAR-EPO. The protein A chip was coupled with anti-VAR mAb (TYJ9-R0016) at 5 µg/mL, while rVAR-EPO concentrations ranged from 100 nM to 6.25 nM in 2-fold dilutions. This anti-VAR mAb shows high affinity for trace amounts of rVAR-EPO. (D) Experimental protocol for reverse–normal immunopurification coupled with western blotting. Eluate from RI and NI were loaded on separate SAR-PAGE gels, followed by western blotting. Ab = antibody; CERA = continuous erythropoietin receptor activator; DP = differential peptide; DYNEPO = epoetin-δ (a type of rEPO); EC50 = 50% effective concentration; EPO = erythropoietin; EPO-Fc = recombinant fusion protein comprising EPO linked to the human immunoglobulin Fc domain; mAb = monoclonal antibody; NESP = darbepoetin-α; NI = normal immunopurification; OD = optical density; pAb = polyclonal antibody; rEPO = recombinant erythropoietin; RI = reverse immunopurification; RT = room temperature; RU = response unit; SAR = sarcosyl; SAR-PAGE = sarcosyl polyacrylamide gel electrophoresis; VAR = variant; VAR-EPO = variant erythropoietin; VAR-PEG = PEGylated recombinant VAR-EPO; WT-EPO = wild-type erythropoietin.

Article Snippet: For NI, 50 μL of the antibody–beads complex was added to the supernatant and incubated with rotation at RT for 1 h. The antibody–beads complex was prepared using 1 μg of anti-EPO polyclonal antibody (AB-286-NA, R&D Systems) and 50 μL of pre-washed magnetic beads, with rotation at 4°C overnight.

Techniques: Immu-Puri, Western Blot, SPR Assay, Concentration Assay, Recombinant, Polyacrylamide Gel Electrophoresis, Variant Assay

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) Localization of the putative pH sensing residues selected for replacement on the three-dimensional structure of the AIP56 catalytic domain. Cartoon (left) and surface representation of AIP56 catalytic domain with (middle) or without (right) the middle domain. The catalytic residues (H165, E166 and H169) are shown in red, the putative pH sensing residues in orange and the D209-K247 hairpin in marine blue. The cysteine residues (pink sticks) forming the disulfide bridge (yellow) are also shown. (B) Analysis of NF-kB p65 cleavage in mouse bone marrow-derived macrophages (mBMDM) by V5 plus His-tagged AIP56 variants. Cleavage of p65 was assessed by western blotting (upper panel; chromogenic detection) and protein loading by staining the membranes with Ponceau S (lower panel). The result shown is representative of six independent experiments. (C) Peak ANS fluorescence measured at 475 nm for the indicated pH, normalized by subtracting the corresponding values at pH 7 (fluorescence due to conformational changes caused by the mutation and not due to acidification). The measurement curves for each pH at different wavelengths are shown in Fig. S7A. The results shown are representative of at least three independent experiments. (D) Coomassie Blue-stained SDS-PAGE gels from limited proteolysis of AIP56 and AIP56 H231K/E234K by Proteinase K. A and B mark the bands corresponding to the catalytic and receptor-binding domains, respectively. (E) Interaction of AIP56 or AIP56 variants with black lipid bilayers. Single channel recordings of DiPhPC/n-decane membranes after addition of the indicated proteins to one side of the black lipid bilayer at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three independent measurements.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Derivative Assay, Western Blot, Staining, Fluorescence, Mutagenesis, SDS Page, Binding Assay, Concentration Assay, Membrane, Activity Assay

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) Schematic representation of chimera Bla L19-W286 AIP56 P210-N497 . (B) FRET-based assay to access the effect of Hsp90 inhibition on Bla delivery. The cleaved/uncleaved CCF4-AM ratios were determined by quantifying a minimum of 10 microscopic fields per condition. Results shown represent one out of three independent experiments. Statistical significance was tested by Kruskal-Wallis nonparametric test and the adjusted p values for individual comparisons were obtained by Bonferroni correction. P values are indicated; ns = non-significant. (C) Control of 17-DMAG activity by confirming its inhibitory effect on NF-kB p65 cleavage upon AIP56 intoxication of mBMDM. A representative blot of three independent experiments is shown. Loading correction was achieved by dividing the density of p65 by the respective density of the Ponceau S staining. Values are mean ± SD.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Inhibition, Control, Activity Assay, Staining

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) V5 plus His-tagged AIP56 modified in the aspartate-rich motif (AIP56 D274S/D276-278S and AIP56 D274N/D276-278N ) is unable to cleave p65 in intact cells. Cleavage of p65 was assessed by western blotting and protein loading by Ponceau S staining. The result shown is representative of six independent experiments. (B) V5 plus His-tagged AIP56 D274S/D276-278S and AIP56 D274N/D276-278N were unable to translocate across the host cell membrane in response to acidification. In all experiments, mock-treated cells were used as controls. NF-kB p65 cleavage was analyzed by western blotting. The result shown is representative of five independent experiments. The box plot shows the quantification of intact NF-kB p65 normalized for Ponceau S. Statistical significance was tested by one-way ANOVA and p values for the individual comparisons were calculated using Tukey’s HSD test. P values are indicated, ns = non-significant. (C) AIP56 D274S/D276-278S and AIP56 D274N/D276-278N retained the ability to interact with black lipid bilayers. Proteins were used at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three independent measurements.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Modification, Western Blot, Staining, Membrane, Concentration Assay, Activity Assay