Detection of free thiols in the enzyme was performed as described previously with some modifications [20] (link). First, protein samples were incubated with 0.25-mM EZ-link iodoacetyl-LC-biotin (Thermo Scientific, IL, USA) for 30 min in the dark and then mixed with 10-mM of dithiothreitol and the × 4 loading buffer. Next, the sample (20-ng protein/lane) was separated by 10% SDS-PAGE and the gels were blotted onto a PVDF membrane; the membrane was then blocked by EzBlock Chemi (ATTO Co., Tokyo, Japan) for 30 min. Finally, the membrane was treated with streptavidin-peroxidase polymer (Merck, ultrasensitive, 1/100,000) for 30 min, and the binding was visualized by Chemi-Lumi One L (Nacalai Tesque, Inc.). This experiment was performed twice.
Ez link iodoacetyl lc biotin
Manufactured by Thermo Fisher Scientific
Sourced in United States
EZ-link iodoacetyl-LC-biotin is a biotin-labeling reagent used for covalent attachment of biotin to free sulfhydryl groups on proteins or peptides. It features a long-chain linker and an iodoacetyl reactive group.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ez link iodoacetyl lc biotin
1
Detection of Free Thiols in Enzyme
2
Free Thiol Detection in Enzymes
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Detection of free thiols in the enzyme was performed as described previously with some modifications [20] (link). First, protein samples were incubated with 0.25-mM EZ-link iodoacetyl-LC-biotin (Thermo Scientific, IL, USA) for 30 min in the dark and then mixed with 10-mM of dithiothreitol and the ×4 loading buffer. Next, the sample (20-ng protein/lane) was separated by 10% SDS-PAGE and the gels were blotted onto a PVDF membrane; the membrane was then blocked by EzBlock Chemi (ATTO Co., Tokyo, Japan) for 30 min. Finally, the membrane was treated with streptavidin-peroxidase polymer (Merck, ultrasensitive, 1/100,000) for 30 min, and the binding was visualized by Chemi-Lumi One L (Nacalai Tesque, Inc.). This experiment was performed twice.
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