Journal: Molecules

Article Title: Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase

doi: 10.3390/molecules24183287

Figure Lengend Snippet: MTGase site specifically labels engineered anti-CD33 mAb. ( a ) Structure of mAb-KF peptide conjugate. Conjugated lysine is highlighted in blue and fluorescein is highlighted in green; ( b ) UV protein stained SDS-PAGE gel of unconjugated mAb and mAb-KF. Molecular weight markers are in kilodaltons (kDa). HC—heavy chain, LC—light chain, HC-KF—heavy-chain-KF conjugate, MTGase—microbial transglutaminase; ( c ) FITC epifluorescence image of the same SDS-PAGE gel. KF—free KF peptide; ( d ) Flow cytometry of mAb-KF stained THP1 (CD33-positive) and Jurkat (CD33-negative) cells. Following spin column cleanup, mAb-KF conjugate was directly diluted in stain buffer at the indicated ratios: blue—1:10,000, green—1:1,000, orange—1:100. Unstained cells are represented in gray. All plots are represented as percent maximum cell count.

Article Snippet: Conjugation efficiency was analyzed by electrophoresis on a 10% SDS-PAGE gel (150 V, 70 min), followed by staining with Blazin’ Bright™ Luminescent UV Protein Gel Stain (Gold Biotechnology, St. Louis, MO, USA) and imaging on a UV transilluminator.

Techniques: Staining, SDS Page, Molecular Weight, Flow Cytometry, Cell Counting

Journal: Molecules

Article Title: Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase

doi: 10.3390/molecules24183287

Figure Lengend Snippet: Conjugation and purification of mAb-KN 3 conjugate. ( a ) Structure of mAb-KN 3 peptide conjugate. Conjugated lysine is highlighted in blue and conjugatable azidolysine is highlighted in red; ( b ) UV protein stained SDS-PAGE gel of unconjugated mAb and mAb-KN 3 . Molecular weight markers are in kilodaltons. HC—heavy chain, LC—light chain, HC-KN 3 —heavy-chain-KN 3 conjugate, MTGase—microbial transglutaminase; ( c ) SEC analysis of the crude mAb-KN 3 conjugation reaction. mAb-KN 3 – soluble mAb-KN 3 conjugate, MTGase—microbial transglutaminase enzyme, Peptide—unconjugated KN 3 peptide. ( d ) SEC purification of mAb-KN 3 conjugate. Gray boxes indicate the elution volumes of collected fractions (6–9).

Article Snippet: Conjugation efficiency was analyzed by electrophoresis on a 10% SDS-PAGE gel (150 V, 70 min), followed by staining with Blazin’ Bright™ Luminescent UV Protein Gel Stain (Gold Biotechnology, St. Louis, MO, USA) and imaging on a UV transilluminator.

Techniques: Conjugation Assay, Purification, Staining, SDS Page, Molecular Weight

Journal: Molecules

Article Title: Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase

doi: 10.3390/molecules24183287

Figure Lengend Snippet: Conjugation and purification of ARC. ( a ) Structure of mAb-KN 3 -DBCO-TEG-siRNA conjugate. Conjugated lysine is highlighted in blue. Conjugated triazole (azidolysine origin) is highlighted in red. Conjugated DBCO moiety is highlighted in green; ( b ) SEC analysis of the crude ARC conjugation reaction. UV absorbance at 260 nm is traced in red and absorbance at 280 nm is traced in blue; ( c ) SEC purification of ARC. Gray boxes indicate the elution volumes of collected fractions (5–7); ( d ) UV protein stained SDS-PAGE gel of unconjugated mAb, mAb-KN 3 and ARC. Molecular weight markers are in kilodaltons. HC—heavy chain, LC—light chain, HC-KN 3 —heavy-chain-KN 3 conjugate, HC-KN 3 -siRNA–heavy-chain-KN 3 -siRNA conjugate.

Article Snippet: Conjugation efficiency was analyzed by electrophoresis on a 10% SDS-PAGE gel (150 V, 70 min), followed by staining with Blazin’ Bright™ Luminescent UV Protein Gel Stain (Gold Biotechnology, St. Louis, MO, USA) and imaging on a UV transilluminator.

Techniques: Conjugation Assay, Purification, Staining, SDS Page, Molecular Weight

Journal: Life Science Alliance

Article Title: Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii

doi: 10.26508/lsa.202201710

Figure Lengend Snippet: (A) Constructs used in this study. Truncated constructs were used where full-length constructs could not be expressed or purified. (A, B) Reactivity of purified constructs from (A) against the activity-based probe Ub–propargylamine (Ub-PA, 1-h reactions). Asterisk (*): Ub-modified construct. (A, C) Reactivity of purified constructs from (A) against activity-based probe NEDD8-PA (1-h reactions). Asterisk (*): NEDD8-modified construct. (D) Cleavage of precursor pro-NEDD8 by TgOTU DUBs. Human DEN1 is used as a positive control. 1-h reactions were resolved on SDS–PAGE, and pro-NEDD8 cleavage was visualised by silver staining. (E) Activity of TgOTU DUBs against the eight diUb linkage types. (A) Purified constructs in (A) at indicated concentrations were incubated with each diUb linkage type (1.2 μM) and sampled at 0′, 5′, and 30′. Samples were resolved on SDS–PAGE, and diUb/monoUb was visualised by silver staining or Lumitein staining. (E, F) Qualitative heatmap summary based on densitometry of cleavage assays in (E). Data are representative of three independent experiments.

Article Snippet: Samples were resolved by SDS–PAGE and visualised with the Silver Stain Plus kit (Bio-Rad) or One-Step Lumitein UV Protein Gel Stain (Biotium).

Techniques: Construct, Purification, Activity Assay, Modification, Positive Control, SDS Page, Silver Staining, Incubation, Staining