free biotin  (Thermo Fisher)

Journal: Journal of Structural Biology: X

Article Title: Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example

doi: 10.1016/j.yjsbx.2025.100126

Figure Lengend Snippet: Workflow of VNAR generation and antigen preparations. (A) Schematic workflow of VNARs generation and application in structural biology. (B) Size-exclusion chromatography purification and SDS-PAGE analysis of CvHAS. The arrow indicates purified CvHAS monomers. (C) Schematic diagram of radioactive signal detection on ascending paper chromatography. (D) Activity Assay of CvHAS. Column 1, reaction with CvHAS and substrates; column 2, reaction without CvHAS; column 3, reaction without one of the substrates, UDP-GA; column 4, reaction product degraded with hyaluronidase; results are shown as the mean ± S.D., n = 3; the asterisks (****) represent p -values ≤ 0.0001. significance of the differences was calculated by paired two-tailed Student’s t -test (the same applies below).

Article Snippet: Excessive biotin was removed from the sample in a biotin-free CvHAS FB buffer using dialysis cups with a 3 kDa cutoff (Thermo Fisher Scientific, USA).

Techniques: Size-exclusion Chromatography, Purification, SDS Page, Paper Chromatography, Activity Assay, Two Tailed Test

Journal: Journal of Structural Biology: X

Article Title: Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example

doi: 10.1016/j.yjsbx.2025.100126

Figure Lengend Snippet: Animal immunization and antigen biotinylation. (A) Schematic diagram of the sites and time schedule of injections. (B) Schematic diagram of biotinylation of CvHAS-Avi-tag. In vitro biotinylation of the protein is catalyzed by the biotin ligase BirA. (C) Validation of CvHAS biotin ligation by Western blotting. M, molecular weight standards (the same applies below), detecting with HRP-linked streptavidin. (D) Assessment of the degree of biotinylation of CvHAS-CA by western blot. His-Tag Mouse mAb was used as detection antibody. The upper arrow indicates the multimer (CvHAS-CA:S.A. molar ratio > 1:1) and the lower arrow indicates that CvHAS-CA and S.A. formed a complex (∼127 kDa) in a 1:1 molar ratio. (E) Activity assay of immobilized CvHAS-CA by different strategies. Results are shown as the mean ± S.D., n = 3; the asterisks (***) represent p -values ≤ 0.001. (F) The ELISA for CvHAS passive adsorption. Results are shown as the mean ± S.D., n = 3; the asterisks (****) represents p -values ≤ 0.0001. (G) ELISA for CvHAS indirect immobilization. Results are shown as the mean ± S.D., n = 3; the asterisks (****) represents p -values ≤ 0.0001. (H) Shark serum IgNAR titer test. Pre-Blood, blood collected before immunization; blood-1 to blood-7, indicating blood collected after each injection; results are shown as the mean ± S.D., n = 3. The schematic diagram indicates the antibodies and interactions used in the titer assay.

Article Snippet: Excessive biotin was removed from the sample in a biotin-free CvHAS FB buffer using dialysis cups with a 3 kDa cutoff (Thermo Fisher Scientific, USA).

Techniques: In Vitro, Biomarker Discovery, Ligation, Western Blot, Molecular Weight, Activity Assay, Enzyme-linked Immunosorbent Assay, Adsorption, Injection, Titer Assay

Journal: Journal of Structural Biology: X

Article Title: Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example

doi: 10.1016/j.yjsbx.2025.100126

Figure Lengend Snippet: Affinity verification and characterization of shark single-domain antibodies. (A) Amino acid sequences and annotations of six selected VNARs. Sequences were analyzed by Jalview software; sequence alignment was performed by the ClustalW algorithm (with default parameters); the red shades indicate cysteines in the CDR1 and CDR3 regions, which form a non-canonical disulfide bond. (B) Clustering of VNAR amino acid sequences after biopanning; regions in different colors indicate amino acid sequences of different clusters. (C) SDS-PAGE analysis of CvHAS and 6 VNARs SEC co-purification elution peaks (related to Fig. S6. C). (D) Determination of VNAR (S2F6) affinity value by isothermal titration calorimetry. (E) Effect of 6 VNARs on the activity of CvHAS respectively. Data are normalized relative to product yields in the absence of VNARs. Results are shown as the mean ± S.D., n = 3.

Article Snippet: Excessive biotin was removed from the sample in a biotin-free CvHAS FB buffer using dialysis cups with a 3 kDa cutoff (Thermo Fisher Scientific, USA).

Techniques: Software, Sequencing, SDS Page, Copurification, Isothermal Titration Calorimetry, Activity Assay

Journal: Journal of Structural Biology: X

Article Title: Generation of shark single-domain antibodies as an aid for Cryo-EM structure determination of membrane proteins: Use hyaluronan synthase as an example

doi: 10.1016/j.yjsbx.2025.100126

Figure Lengend Snippet: The structure of CvHAS-VNAR complex. (A) Schematic diagram of the assembly of CvHAS-VNAR complex. (B) CvHAS-VNAR complex structure (PDB: 9KQC). CvHAS and VNAR were colored with green and cyan, respectively. The interaction residues at the binding interface between CvHAS and VNAR are expanded. CH-π interactions include CvHAS (LEU218) − VNAR (TRP93), and CvHAS (HIS195) − VNAR (ILE95). Hydrogen bonds include CvHAS (HIS195) − VNAR (PHE96), CvHAS (PRO222) − VNAR (ASN94), and CvHAS (SER219) − VNAR (CYS92).

Article Snippet: Excessive biotin was removed from the sample in a biotin-free CvHAS FB buffer using dialysis cups with a 3 kDa cutoff (Thermo Fisher Scientific, USA).

Techniques: Binding Assay