The use of Ni-nitrilotriacetic acid agarose for estimation of affinities of hexahistidine-tagged Fab to single-stranded DNA.
The use of Ni-nitrilotriacetic acid agarose for estimation of affinities of hexahistidine-tagged Fab to single-stranded DNA.
复制标题
使用 Ni-次氮基三乙酸琼脂糖估计六组氨酸标记的 Fab 与单链 DNA 的亲和力。
DOI：
10.1006/abio.1997.2051
复制
发表时间：
1997
影响因子：
2.9
通讯作者：
Deutscher,SL
中科院分区：
文献类型：
--
作者：
Komissarov,AA;Marchbank,MT;Deutscher,SL
32 P标记的（dT）15和六组氨酸（6-His）标记的抗单链DNA（ssDNA）Fab（DNA-1）之间形成的复合物通过加入镍螯合的次氮基三乙酸（Ni-NTA）琼脂糖来捕获，从而有效地分离结合的配体和游离的配体。固定化复合物的高稳定性（半衰期为4 h）和[32 P]（dT）15的低非特异性结合允许快速估计解离常数（Kd），并发现其为约130 nm。寡核苷酸结合的DNA-1与溶液中形成的Fab/（dT）15复合物具有相同的K值，表明6-His标签与树脂的相互作用不干扰结合。加入未标记的（dT）15导致与结合的[32 P]（dT）15的快速交换。还检查了DNA-1的突变形式，所得结果与平衡凝胶过滤和荧光滴定的数据一致[A. A. Komissarov，M.J. Calcutt，M.T. Marchbank，E.N. Peletskaya和S.L. Deutscher（1996）J.Biol.Chem.271，12241-12246]。这些结果表明，Ni-NTA测定是一种有效和准确的方法来检查6-His-标记的蛋白质-核酸复合物。此外，该测定的竞争性修改可用于检测血清中的抗ssDNA抗体。
The complex formed between32P-labeled (dT)15and a hexahistidine (6-His)-tagged anti-single-stranded DNA (ssDNA) Fab, DNA-1, was trapped by addition of nickel-chelating nitrilotriacetic acid (Ni–NTA) agarose that led to efficient separation of bound ligand from free. High stability of the immobilized complex (half-life of 4 h) and low nonspecific binding of [32P](dT)15allowed for a rapid estimation of the dissociation constant (Kd) and was found to be ∼130 nm. Oligonucleotide bound DNA-1 preimmobilized on Ni–NTA agarose with the sameKdas the Fab/(dT)15complex formed in solution, indicating that the interaction of the 6-His tag with the resin did not interfere with binding. Addition of unlabeled (dT)15led to a fast exchange with bound [32P](dT)15. Mutant versions of DNA-1 were also examined and results obtained were in agreement with data from equilibrium gel filtration and fluorescence titration [A. A. Komissarov, M.J. Calcutt, M.T. Marchbank, E.N. Peletskaya, and S.L. Deutscher (1996)J. Biol. Chem.271, 12241–12246]. These results demonstrate that the Ni–NTA assay is an efficient and accurate method to examine 6-His-tagged protein–nucleic acid complexes. Furthermore, a competition modification of this assay may be used for detection of anti-ssDNA antibodies in serum.