mouse anti his tag ab  (SouthernBiotech)

Journal: Molecular immunology

Article Title: Anti-tumor effectiveness of a novel bispecific antibody that blocks both PD-L1 and LAG-3.

doi: 10.1016/j.molimm.2025.03.015

Figure Lengend Snippet: Fig. 2. Cell-targeting activities of scFv and Ba-PL. (A) Western blot analysis of PD-L1 expression on tumor cells. H22, 4T1, CT26 and B16 cells were precultured in RPMI 1640 medium with 100 ng/mL IFN-γ for 72 h. Cells were lysed using RIPA buffer, and the supernatant was resolved by SDS-PAGE. After centrifugation, the samples were transferred to PVDF membranes for Western blotting. (B) Representative flow cytometry histograms showing the binding of single-chain variable fragment (scFv) and bispecific antibody (BsAb) to CT26, B16, 4T1, and H22 cells, as well as lymphocytes. The fluorescence signal shifts indicate the binding activity of BsAb and scFv to the target cells. Anti-PD-L1 antibody (Ab) and anti-LAG-3 Ab served as positive controls, while mIgG was used as a negative control. Binding of Ba-PL and scFv-PD-L1 to PD-L1 expressed on tumor cells was detected using an FITC-conjugated anti-His tag mouse monoclonal antibody (mAb). Binding of Ba-PL and scFv-LAG-3 to LAG-3 expressed on lymphocytes was detected using a PC5.5-conjugated anti-His tag mouse mAb. (C, D) Confocal laser microscopy images showing the binding of Ba-PL to tumor cells and lymphocytes. Cells were stained with DAPI for nuclei, FITC for PD-L1 binding, and PC5.5 for LAG-3 binding. Scale bars: 50 μm.

Article Snippet: Following washing with PBS, the fluorescein isothiocyanate (FITC)conjugated mouse anti-His tag mAb (5 μg/mL; Bioss) was added and incubated for another 1 h in the dark at 4 °C.

Techniques: Western Blot, Expressing, SDS Page, Centrifugation, Flow Cytometry, Binding Assay, Fluorescence, Activity Assay, Negative Control, Microscopy, Staining

Journal: medRxiv

Article Title: Automated Cell-free Protein Synthesis for Distributed Biomanufacturing

doi: 10.1101/2025.10.15.25338083

Figure Lengend Snippet: (a) Nanobodies targeting the SARS-CoV-2 spike protein and murine TNF, as well as the TNF itself, were produced on MANGO from CFPS reactions (b) After production, the products, including TNF (predominantly monomer with traces of dimer), TNF Nb, and anti-spike Nb (Nb21), were analyzed using 4-20% gradient SDS-PAGE and stained with ProBlue Safe, which confirmed their presence at the correct size. The molecular weight ladder (in kilodaltons) is shown on the left. (c) ELISA was performed against recombinant spike protein receptor-binding domain (RBD) using MANGO-produced anti-spike Nb21 as the primary antibody and HRP-conjugated anti-His₆ antibody as the secondary. Detection was performed using the QuantaBlu fluorogenic peroxidase substrate kit, and fluorescence intensity was measured in a conventional plate reader. (d) TNF neutralization assay was conducted using anti-TNF Nb (MANGO) and L929 cells, demonstrating (e) cell survival after preincubation of TNF (1 ng/mL) with both in-house and commercial antibodies. (f) Dose-response curves in the presence or absence of MANGO anti-TNF Nb. Luminescence was measured with a standard plate reader. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Data are shown as the mean of three technical replicates ± SD (ns p-value > 0.05, **** p-value < 0.0001). Abbreviations: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; Nb, nanobody.

Article Snippet: After six additional washes with PBST, wells were incubated for 1 hour at 350 rpm with anti-His6-HRP Ab (SouthernBiotech, 4603-05, 1:6,000) prepared in 0.5% skim milk (Bioshop) in PBST (Gibco).

Techniques: Produced, SDS Page, Staining, Molecular Weight, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Fluorescence, Neutralization