Hispur ni nta resin

Manufactured by GE Healthcare

Sourced in China, United States

HisPur™ Ni-NTA Resin is a nickel-nitrilotriacetic acid (Ni-NTA) agarose resin designed for the purification of His-tagged proteins. The resin utilizes the high-affinity interaction between the histidine tag and the immobilized nickel ions to selectively capture target proteins from complex mixtures.

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Lab products found in correlation

Superdex 75, by GE Healthcare (1 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions) Ion exchange column, by GE Healthcare (1 mentions) Glutathione sepharose 4b manual, by GE Healthcare (1 mentions) Mc lr, by Enzo Life Sciences (1 mentions) M13k07 helper phage, by New England Biolabs (1 mentions)

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Ni-NTA resin (63 citations) Ni-NTA (45 citations)

5 protocols using hispur ni nta resin

Purification of Recombinant Proteins

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After centrifugation, the supernatant was loaded onto a column containing HisPur™ Ni-NTA Resin (GE Healthcare) for His-tag affinity purification. The column was washed five times with wash buffer (50 mM Tris, 300 mM NaCl, 20% glycerol, 50 mM imidazole, pH 7.5) to remove contaminating proteins. The target protein was eluted with elution buffer (50 mM Tris, 300 mM NaCl, 20% glycerol, 500 mM imidazole, pH 7.5). The finally obtained protein was analyzed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
FDR2 was further purified by size-exclusion chromatography at 10−20 °C on gel filtration column (GE Healthcare, Superdex 75), in three protein loads on a column. Then FDX1 was further purified by ion exchange column (GE Healthcare) with buffer A (20 mM MES, 20% glycerol, pH 6.5) and buffer B (20 mM MES, 1M NaCl, 20% glycerol, pH 6.5), in two proteins loads on a column. Purified fractions were checked on SDS-PAGE gel. Protein concentrations were measured with NanoDrop Spectrophotometer (Thermo Scientific) at 280 nm. Purified proteins were snap-frozen in liquid nitrogen and stored at −80 °C.

Yang L., Yang D., Wang Q., Li J., Li H.L, & Pan L. (2022). Functional expression and purification of DoxA, a key cytochrome P450 from Streptomyces peucetius ATCC 27952. PeerJ, 10, e14373.

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Affinity Purification of Protein Interactions

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To determine protein-protein interactions, the His-tagged proteins were respectively bound to 50 μl HisPur Ni-NTA Resin (GE Healthcare) and then incubated with GST-tagged proteins at 4 °C for 1 h. After washed three times with the NET/NP-40 buffer at 4 °C, the beads were eluted with 250 mM Imidazole and then subjected to western blot analysis using both anti-GST and anti-His antibodies.

Zhang W., Cai C., Lin L., Tao Y.J, & Jin M. (2017). Subcellular localization and interactions of Infectious Salmon Anemia Virus (ISAV) M1 and NEP as well as host Hsc70. Virology Journal, 14, 30.

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Purification of His-tagged and GST-tagged Proteins

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The expression of His-M1, His-Hsc70, GST-Hsc70, and GST-NEP were induced with 1 mM IPTG at 15 °C for 20 h after the cell density reached an OD₆₀₀ nm of 0.6. Cells were collected and sonicated in a lysis buffer [300 mM NaCl, 5 mM Imidazole, 10% (v/v) glycerol, and 50 mM Tris · HCl (pH 7.5)]. The lysate was centrifuged at 12,000 rpm for 40 min, and the supernatant was used for further purification. The His-tagged proteins were purified using HisPur Ni-NTA Resin (GE Healthcare), while GST-tagged proteins were purified using glutathione sepharose 4B manual (GE Healthcare). All purified proteins were dialyzed in a NET/NP-40 buffer [50 mM Tris · HCl (pH 7.9), 0.1 M NaCl, 5 mM EDTA, and 0.1% NP-40], which was also used as wash buffer.

Zhang W., Cai C., Lin L., Tao Y.J, & Jin M. (2017). Subcellular localization and interactions of Infectious Salmon Anemia Virus (ISAV) M1 and NEP as well as host Hsc70. Virology Journal, 14, 30.

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Nanobody-based ELISA for Cyanotoxin Detection

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Nodularin-R and microcystins (MC-LR, -LA, -LY, -LW, -LF, -YR, -WR, -RR) (Enzo, USA) were used as standards. Nodularin-R-ovalbumin (NOD-R-OVA, prepared in our lab) was the coating antigen. Microcystin-LR-keyhole limpet hemocyanin (MC-LR-KLH, prepared in our lab) was used as the immunogen. An anti-MC-LR monoclonal antibody (anti-MC-LR MAb, prepared in our lab) was used for comparison with the nanobodies. pComb3XSS vector and E. coli BL21(DE3) available in our lab were used to construct a recombinant vector and a transformed host strain, respectively. Helper phage M13K07 (New England Biolabs, Ipswich, MA, USA) was used for the construction of a phage displayed nanobody library. The anti-VHH-HRP polyclonal antibody from rabbit (Genscript Bio. Co.Ltd, Nanjing, China) was used as a secondary antibody in ELISA. HisPur Ni-NTA resin (GE Healthcare, Beijing, China) was utilized for protein purification. The primers used in this work were synthesized by Invitrogen Biotechnology Co. (Shanghai, China). Other chemical reagents were provided by Sigma (St. Louis, MS, USA) and Thermo Fisher Scientific (Thermo, Waltham, MA, USA).

Yang J., Si R., Wu G., Wang Y., Fang R., Liu F., Wang F., Lei H., Shen Y., Zhang Q, & Wang H. (2021). Preparation of Specific Nanobodies and Their Application in the Rapid Detection of Nodularin-R in Water Samples. Foods, 10(11), 2758.

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His-tag Affinity Purification of Recombinant Proteins

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After centrifugation, the supernatant was loaded onto a column containing HisPur™ Ni-NTA Resin (GE Healthcare, Chicago, IL, USA) for His-tag affinity purification. The column was washed five times with wash buffer (50 mM Tris, 300 mM NaCl, 10% glycerol and 50 mM imidazole at pH 7.5) to remove contaminating proteins. The target protein was eluted with elution buffer (50 mM Tris, 300 mM NaCl, 10% glycerol and 500 mM imidazole at pH 7.5). The finally obtained protein was analyzed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Yang L., Zhou H., Chen G., Li H., Yang D, & Pan L. (2023). Expression and Purification of Glycosyltransferase DnmS from Streptomyces peucetius ATCC 27952 and Study on Catalytic Characterization of Its Reverse Glycosyltransferase Reaction. Microorganisms, 11(3), 762.

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