Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Schematic of a DNA sequence map of hIL7 expression vectors.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Sequencing, Expressing

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Purification and identification of the fusion protein. (a) Transformation of the recombinant plasmid into competent cells, plated on LB solid medium supplemented with ampicillin. (b) SDS-PAGE with Coomassie blue staining of proteins extracted from recombinant bacteria cultured in LB liquid medium containing ampicillin at 37 °C and 200 rpm under different culture durations (4, 8, 12 h) and different IPTG concentrations (0.1, 0.5, 1.0 mM). (c) Analysis of protein expression under uninduced conditions and after induction (12 h, 0.5 mM IPTG) in LB liquid medium with ampicillin at 37 °C and 200 rpm. Samples include uninduced, pellet, supernatant, flow-through, and elution fractions with 300 mM imidazole. The right panel shows the purification of soluble proteins and Western blot detection using an anti-His antibody. (d) SDS-PAGE with Coomassie blue staining of soluble and inclusion body proteins. Lanes from left to right represent pellet, supernatant, flow-through, and elution fractions with different imidazole concentrations. (E)­Western blot analysis of soluble and inclusion body proteins using anti-hIL7 and antibiotin antibodies is also shown. Note: the following terms are used throughout the experimental procedures. Pellet: solid fraction obtained after centrifugation. Supernatant: liquid fraction obtained after centrifugation. Flow-through: fraction that passes through the His-tag nickel affinity column during sample loading. Washing: fraction eluted with a low concentration imidazole (or ionic) buffer to remove nonspecifically bound impurities. Elution: fraction containing target protein, eluted with high concentration imidazole (or ionic) buffer for competitive displacement. Concentration notation: mM = millimolar concentration. Notation 300 mM-1 refers to first elution step performed with 300 mM imidazole and subsequent elutions are labeled accordingly (e.g., 300 mM-2, 300 mM-3).

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Purification, Transformation Assay, Recombinant, Plasmid Preparation, SDS Page, Staining, Bacteria, Cell Culture, Expressing, Western Blot, Centrifugation, Affinity Column, Concentration Assay, Labeling

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Extraction and stepwise elution of inclusion body proteins. (a) Western blot analysis of fractions from the solubilization and washing of inclusion bodies under pH 4.6 or 250 mM imidazole conditions. Lanes (left to right): supernatant, flow-through, wash, and elute fractions for each condition. The membrane was probed with anti-hIL7 and antibiotin antibodies. (b) Coomassie blue-stained SDS-PAGE analysis of proteins during the washing and elution processes. Lanes show the supernatant, flow-through, and sequentially washed fractions by low concentration gradients of imidazole (six washes each at 10, 20, and 30 mM imidazole), followed by ten consecutive elutions using 250 mM imidazole. Note that “mM” indicates millimolar; “10 mM-2” is the second wash with 10 mM imidazole, and subsequent steps are labeled in the same manner.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Extraction, Western Blot, Membrane, Staining, SDS Page, Concentration Assay, Labeling

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Functional validation of the fusion protein. (a) Western blot analysis probed with anti-hIL7 antibody and SA-HRP to evaluate biotinylation efficiency in the presence or absence of exogenous biotin supplementation. (b) Analysis of the conjugation between randomly biotinylated Bovine Serum Albumin (BSA) and SA by native page. Lanes represent different concentrations of SA and BSA. Additionally, the conjugation efficiency of site-specifically biotinylated Avi-tagged Biotin-IL15 and the fusion protein biotin-IL7 with SA are compared. Note: um: Micromolar concentration (c) CCK-8 assay assessing the proliferation of preactivated T cells after coculture with the fusion protein that had been desalted and concentrated via ultrafiltration.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Functional Assay, Biomarker Discovery, Western Blot, Conjugation Assay, Clear Native PAGE, Concentration Assay, CCK-8 Assay