Cleavage assay was performed as we have previously described using recombinant His tagged VWF-A2 protein with modifications [12 (link)]. Briefly, A2 protein subjected to in vitro FAM20c kinase assay in the presence or absence of ATP was absorbed to Ni-NTA HisSorb strips (Qiagen) as substrate. Microtiter wells were incubated with diluted plasma for 16 hours. Following washing, A2 was detected with human VWF-A2 antibody (R&D Systems, Clone # 210909), which recognized amino acids 1606–1665 C terminus of the cleavage site. Cleavage of A2 by plasma was shown as reduction of VWF-A2 antibody recognition as compared to TBS control.
Ni nta hissorb strips
Manufactured by Qiagen
The Ni-NTA HisSorb strips are a laboratory equipment product designed for the purification of histidine-tagged proteins. The strips are pre-coated with Ni-NTA (Nickel-Nitrilotriacetic Acid) resin, which selectively binds to the histidine-tagged proteins. This allows for the efficient capture and purification of the target proteins from complex mixtures.
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2 protocols using ni nta hissorb strips
1
VWF-A2 Cleavage Assay Protocol
2
Quantitative Strep-Tag Protein Assay
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ELISA was performed in Ni-NTA HisSorb Strips (Qiagen, Hilden, DE) according the manufacturer instructions. A 1:5,000 dilution of monoclonal anti-Strep-tag II antibodies conjugated to HRP (IBA, Goettingen, DE), the 1-Step Slow TMB-ELISA substrate and Stop Solution (Thermo Scientific, Waltham, MA, US) were used for colorometric detection at OD450nm after 30 min of incubation. All reactions were performed in triplicate and wells without Dip, without RNA degradosome or without both proteins were used as a negative control.
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