Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link® (ab201795)

Direct ELISA dose response curve generated with varying concentration of biotin conjugated antibodies. Ab242835, ab242990, ab242955 and ab242950 were conjugated to biotin using Biotin Conjugation Kit (Fast, Type A) - Lightning-Link® (ab201795) to produce Conjugate A, B, C and Control respectively. Briefly, each well was coated with Recombinant Protein G.  After blocking with 5% BSA blocking buffer for 2 hours, plates were coated with 1ug/ml of biotin conjugated antibodies. Detection was performed using a HRP-conjugated Streptavidin (ab7403),  and visualised using a TMB substrate. Plates were read at 450nm using an Agilent BioTek Synergy HTX multimode reader.

Drachsler, Moritz, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining expression of CD95. They used the kit to conjugate biotin to anti-human CD95 for use in Western blot.
IP for CD95 and P-Tyrosine GSCs in naive, CD95-WT and CD95-mut GBM cells stimulated with CD95L-T4. Blots were probed with anti-P85 (regulatory PI3K subunit), anti-Sfk, anti-CD95 or anti-P-Tyrosine antibodies, respectively.

Hähnel, Viola, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining cytokine levels of plasma donors. They used the kit to conjugate Biotinylation Kit / Biotin to cytokine proteins for use in multiplex protein detection.
Cytokines, lipopolysaccharide binding protein (LBP) and bactericidal permeability increasing protein (BPI) of convalescent donors were compared to healthy control group. The following cytokines were below limit of detection: IL1β, IL1RA, IL2, IL3, IL6, IL10, IL12p40, IL15, IL21, IL22, IL23, IFNβ, IFNγ, GM-CSF, MIP1α and TNFα.

Hansen, Uwe, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining WARP-collagen IV interactions in in vitro in cartilage. They used the kit to conjugate biotin to recombinant WARP for use in Electron Microscopy.
Biotinylated recombinant WARP was visualized by 5 nm gold labeled with streptavidin. Scale bar is 25 nm

Soleto, Irene, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining the effect of APRIL on B cells using rainbow trout (Oncorhynchus mykiss) as a model species. They used the kit to conjugate biotin to a recombinant trout proliferation-inducing ligand (APRIL) for use in Fluorescence microscopy.
Total leukocytes from spleen were incubated with recombinant biotinylated APRIL (1 µg/ml) for 15 min, then plated onto poly-l-lysine-coated glass slides, fixed and labeled with anti-IgM (shown as red) and FITC-streptavidin (APRIL, shown as green), then counterstained with DAPI (blue), and analyzed by confocal fluorescence microscopy.

Liu, Chien-Chun, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining assays for snakebite detection based on clinical antivenom usage. They used the kit to conjugate biotin to neurotoxic species-specific antibodies (NSS-Abs) and hemorrhagic species-specific antibodies (HSS-Abs) for use in ELISA.
(A, B) Venom proteins of (A) Trimeresurus stejnegeri and (B) Protobothrops mucrosquamatus were diluted in human plasma (10 ng venom protein per ml of plasma) and then mixed with serially diluted Freeze-dried hemorrhagic antivenom (FHAV) (0.32 to 1000 nl) at room temperature for 30 min. The mixtures were then subjected to HSS-Ab–based sandwich ELISA assay. (C, D) Venom proteins of (C) Bungarus multicinctus and (D) Naja atra were diluted in human plasma (10 ng venom protein per ml of plasma) and then mixed with serially diluted FHAV (0.32 to 1000 nl) at room temperature for 30 min. The mixtures were then subjected to NSS-Ab–based sandwich ELISA assay.

He, Xiaohua, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining the detection of abrin by monoclonal antibodies. They used the kit to conjugate biotin to anti-Abrin antibody for use in ELISA.
Detection of abrin in PBS by ELISA. Data represent the average of three determinations ± SD. Limit of detection (LOD) for the mixture of abrin was 1 ng/mL. The one-sided 95% confidence intervals on the fitted line are shown as dashed curves.

Giussani, Stefania, et al used Biotinylation Kit / Biotin Conjugation Kit (Fast, Type A) - Lightning-Link^® (ab201795) as part of examining interaction of C3d ligand with complement interfering protein (CIP). They used the kit to conjugate biotin to C3d for use in flow cytometry.
Flow cytometry analysis of C3d interaction with B cells in presence or absence of Complement Interfering Protein (CIP). A) Biotinylated C3d was preincubated with SA and then with 1–9 μM of CIP, 9 μM of Fib3, or buffer alone. Each mixture was added to Raji B cells and treated as described. Binding of the biotinylated C3d–SA complex to cells was revealed by flow cytometry using a C3d-specific mAb and a phycoerythrin-labeled secondary antibody. Mean fluorescence intensity values of the peaks were analyzed by the FlowJo software. The graph shows the percentage of the residual mean fluorescence intensity values in the presence of a competitor compared with buffer alone, as derived from 4 independent experiments. B) Flow cytometry analysis of C3d binding to enriched B cells from human PBMCs in presence or absence of 1.5 or 9 μM CIP or 9 μM of Fib3; experimental conditions were the same as in A.

This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.

Liu T et al. used ab201795 as part of examining glycobiomarker in hepatocellular carcinoma metastasis.

They used the kit to conjugate biotin to total proteins from lysate for use in a lectin microarray.

Lectin microarray analysis of glycoforms. (A) The lectin microarray contains 50 lectin spots with different binding specificities. (B) Scan image of the lectin microarray incubated with biotinylated proteins and Cy5 labeled streptavidin. Typical glycan profiles of HCC with metastasis were shown on the right and HCC with non-metastasis on the left. (C) Hierarchical clustering of positive lectin binding spots (S/B≥2). Each row represented a single lectin, S/B values were shown by the color scale: red represents a lectin with high S/B value while green represents a lectin with low S/B value.