AMG655 (Conatumumab) was a kind gift from Amgen. This fully humanised antibody, which recognises the extracellular region of DR5, was conjugated to Dynabeads® using the antibody coupling kit from Invitrogen as per manufacturer’s instructions. For the DR5 DISC assay, 30μL of Dynabeads® coated with 5μg of AMG655 was added to 2×106 cells and incubated for 1hr. The cells were then lysed in DISC buffer (0.2% NP-40, 20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 10% glycerol). The AMG655-coated Dynabeads® were collected and washed 5 times in DISC buffer prior to resuspension in Laemmli buffer and analysis by Western blotting. Supernatants and inputs were also collected and analysed by Western blotting.
Antibody coupling kit
Manufactured by Thermo Fisher Scientific
The Antibody Coupling Kit is a laboratory tool used to covalently link antibodies to various surfaces or substrates, such as beads or microplates. The kit provides the necessary reagents and protocols to facilitate this antibody immobilization process, which is essential for many immunoassay and affinity-based applications.
Automatically generated - may contain errors
6 protocols using antibody coupling kit
1
DR5-Mediated Death-Inducing Signaling Complex Assay
2
Exploring Protein Interactions via Co-IP
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Co-immunoprecipitation (Co-IP) was performed to explore the protein interactions on the targeted protein using an Antibody Coupling Kit (#14311D, Invitrogen) as previously reported [24 (link)]. The dynabeads were incubated with the specified antibodies for 16–24 h accordingly, and then incubated with the cell lysates extracted from HCC cells for 1–2 h. The protein-dynabeads-specified antibodies complexes were mixed with blue SDS-binding buffer, and subjected to the incubation in 70 °C water for 10 min. Subsequently, interacting proteins were separated from the complexes under the centrifugation at 13,000 rpm for 2 min. After that, the supernatant was used for further analysis, including biological mass spectrometry and western blotting.
3
Protein Interaction Detection by Co-IP Analysis
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Protein interaction was detected by co‐IP analysis with an Antibody Coupling Kit (Invitrogen). Dynabeads were used to couple the specific antibodies, including STUB1, HSP90β, and YTHDF2, with incubation for 16–24 h. Cell lysates isolated from HCC or HEK293T cells were incubated with Dynabeads‐coupled antibodies. Next, SDS buffer was added to the mixture containing protein‐Dynabeads‐antibodies, followed by incubation at 70 °C for 10 min. Finally, the targeted/combined proteins were isolated from the mixtures via centrifugation. The supernatant was used for further LC‐MS/MS analysis or western blotting, a previously described routine assay.[10 ]
4
DR5-Mediated Death-Inducing Signaling Complex Assay
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AMG655 (Conatumumab) was a kind gift from Amgen. This fully humanised antibody, which recognises the extracellular region of DR5, was conjugated to Dynabeads® using the antibody coupling kit from Invitrogen as per manufacturer’s instructions. For the DR5 DISC assay, 30μL of Dynabeads® coated with 5μg of AMG655 was added to 2×106 cells and incubated for 1hr. The cells were then lysed in DISC buffer (0.2% NP-40, 20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 10% glycerol). The AMG655-coated Dynabeads® were collected and washed 5 times in DISC buffer prior to resuspension in Laemmli buffer and analysis by Western blotting. Supernatants and inputs were also collected and analysed by Western blotting.
5
Proteomic Analysis of Gamete Interactome
6
Immunoprecipitation and Mass Spectrometry for GSAP Interactors
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HA or GSAP antibody was covalently conjugated to Dynabeads M-270 Epoxy beads (#14301; Thermo Fisher Scientific) using the antibody coupling kit (#14311D; Thermo Fisher Scientific). Cells cultured in triplicate were lysed in 1% CHAPSO IP lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, supplemented with protease inhibitor cocktail, and PhosStop) and diluted in IP lysis buffer to reach a CHAPSO final concentration of 0.25%. HA or GSAP antibody-conjugated beads were added into the lysate to tumble for 2 h at 4°C. Magnetic beads were then collected and washed three times with 0.25% CHAPSO IP lysis buffer and three times with PBS. The immunoprecipitates were eluted with 8 M urea. Proteins were digested overnight with Endopeptidase Lys-C and trypsin. Peptides were analyzed by nano–liquid chromatography (nano-LC) MS/MS. Data were processed using MaxQuant. Comparing bait versus control samples, a differentially enriched protein was labeled as a GSAP-binding protein candidate when it had either an average difference >1.5 or P value < 0.05. Candidates were subjected to GO biological pathway analysis using DAVID 6.8 (https://david.ncifcrf.gov/ ). Meta-enrichment analyses were performed using Metascape (Zhou et al., 2019 (link)).
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