recombinant human ca125 muc16 protein  (R&D Systems)

Journal: Cancers

Article Title: Humanized Anti-MUC16 Antibody-Conjugated Contrast Agents for Magnetic Resonance Imaging of Pancreatic Cancer

doi: 10.3390/cancers17060957

Figure Lengend Snippet: Targeting MRI conjugate. ( a ) Immunofluorescence (IF) staining for MUC16 using huAR9.6 on T3 M4 WT (MUC16+) and T3 M4 MUC16 KO isogenic PDAC cell lines; ( b ) synthesis of the antibody conjugate for MRI via conjugation of huAR9.6 and huIgG (isotype control) with DTPA and gadolinium chloride to yield antibody–Gd–DTPA; ( c ) the spectrometric distribution (300 nm to 700 nm) of standard Gd chloride, huIgG–Gd-DTPA and huAR9.6–Gd–DTPA conjugates; ( d ) enzyme-linked immunosorbent assay (ELIZA) depicting huAR9.6 and huAR9.6–Gd–DTPA binding affinity to MUC16 TR 1.2 ( n = 3).

Article Snippet: Additionally, an ELISA (Enzyme-linked immunosorbent assay) was performed as follows: a recombinant MUC16 fragment (Trx-1.2 T, 20 μg/mL in PBS) was immobilized in the wells of MaxiSorp microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) and blocked overnight (4 °C) with bovine serum albumin (1% in PBS) as previously described (19).

Techniques: Immunofluorescence, Staining, Conjugation Assay, Control, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Nature biotechnology

Article Title: Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

doi: 10.1038/s41587-023-01840-6

Figure Lengend Snippet: a, Digestion of recombinant human MUC16 (rhMUC16) with eStcE alone or nanobody-eStcE conjugates. b, rhMUC16 in-gel digest depicting degradation of eStcE-αHER2 conjugate after long-term storage at 4 °C. c, Representative flow plots showing cell surface binding of nanobody alone and eStcE-αHER2 on MCF10AHER2 cells measured by anti-His staining. d, Cell surface binding curves derived from mean fluorescence intensity from (c) (n = 3 biologically independent replicates). e, Kd values derived from (d). f, Representative flow plots showing cell surface binding of αHER2-eStcE on MCF10A±MUC1, ±HER2 cells measured by anti-His staining. Flow plot of αHER2-eStcE on MCF10AHER2 is shown in (c). g, Kd values derived from mean fluorescence intensity from (f) and Fig. 3c (n = 3 biologically independent replicates). The MCF10AHER2 bar depicts the same data as the αHER2-eStcE bar in (e). Data are mean ± s.d. P-values were determined using Tukey-corrected one-way ANOVA. *p < 0.05, **p < 0.005, ***p < 0.0005.

Article Snippet: For recombinant human MUC16 digestion experiments, recombinant MUC16 (R&D Systems) was left unlabeled but was otherwise treated as described above.

Techniques: Recombinant, Binding Assay, Staining, Derivative Assay, Fluorescence

Journal: Nature biotechnology

Article Title: Design of a mucin-selective protease for targeted degradation of cancer-associated mucins

doi: 10.1038/s41587-023-01840-6

Figure Lengend Snippet: a-b, 4T07MUC1 cells (a) and OVCAR-3 cells (b) were treated with 50 nM StcE for 2 hours, washed 1x with 2 mM EDTA followed by 5x with DPBS, then cultured for the indicated times. Cells were then lysed and subjected to Western blotting for MUC1 (a) and MUC16 (b). Mucin bands are denoted by black arrows. c, Bioluminescent imaging of animals described in Fig. 4e. d, Total flux measurements quantified from (c). e, Plot depicting mouse masses of animals described in Fig. 4e. f-g, H&E staining of lungs from PBS-treated (f) or αHER2-eStcE treated (g) animals (n = 7 animals per group, 2 slides per animal). Percent area of lung metastases is quantified in Fig. 4g. h, Quantification of images from Supplementary Fig. 6 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the cytosol. i, Quantification of images from Supplementary Fig. 7 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the cytosol. j, Quantification of images from Supplementary Fig. 8 using the IHC profiler plugin in ImageJ. Percent positive corresponds to positive DAB staining in the nucleus. k, Treatment regimen for BALB/c mice injected intravenously (I.V.) via tail vein with 4T07MUC1, HER2 cells. Doxycycline was included in the chow for the duration of the experiment to maintain MUC1 ectodomain expression. αHER2-eStcE at 10 mg/kg or an equimolar quantity of αHER2-eStcEE447D or αGFP-eStcE were injected I.V. every other day starting on day 0 (n = 9 animals per group). l, Total flux of the indicated days normalized to the total flux on day 0 for each mouse quantified from Supplementary Fig. 10. Data are mean ± s.e.m. P-values were determined using two-tailed Mann-Whitney test (d-e), two-tailed unpaired t-test (h-j), or Tukey-corrected one-way ANOVA (l). *p < 0.05, **p < 0.005, ***p < 0.0005.

Article Snippet: For recombinant human MUC16 digestion experiments, recombinant MUC16 (R&D Systems) was left unlabeled but was otherwise treated as described above.

Techniques: Cell Culture, Western Blot, Imaging, Staining, Injection, Expressing, Two Tailed Test, MANN-WHITNEY