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Ni nta his binding resin

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Ni-NTA His binding resin is a nickel-charged resin used for the purification of histidine-tagged proteins. It provides a simple and efficient method for the capture and elution of recombinant proteins expressed with a polyhistidine tag.

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Lab products found in correlation

Glutathione agarose, by Thermo Fisher Scientific (2 mentions) Coomassie brilliant blue staining, by Bio-Rad (2 mentions) Fla 3000 plus dage system, by Fujifilm (2 mentions) γ 32p atp, by PerkinElmer (1 mentions) Neg502a001mc, by PerkinElmer (1 mentions) Phosstop, by Roche (1 mentions) Kta pure chromatography system, by GE Healthcare (1 mentions) Superdex 200 increase column, by GE Healthcare (1 mentions) Monoclonal anti gst, by Cell Signaling Technology (1 mentions) Glutathione sepharose 4b gst tagged protein purification resin, by GE Healthcare (1 mentions) Quick start bradford reagent, by Bio-Rad (1 mentions) Bio safe coomassie stain, by Bio-Rad (1 mentions)

7 protocols using ni nta his binding resin

1

Purification and In Vitro Kinase Assay

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To express recombinant GST-MEL1 and His-PID proteins, the respective constructs were transformed into E. coli BL21 (DE3) cells for protein expression. Cultures at OD600 of 0.6 were induced with 0.5mM IPTG at 16°C overnight. Proteins were purified using Glutathione agarose for GST-MEL1 and Ni-NTA His binding resin for His-PID following the manufacturer’s instructions (Thermo Scientific). Purified proteins were analysed by SDS-PAGE and visualized by Coomassie brilliant blue staining (Bio-Rad). In vitro protein kinase assay with [γ-32P] ATP was carried out as previously reported with minor modifications 43 (link). Recombinant GST-MEL1 (2 μg) and His-PID (5 μg) proteins were incubated together in 25 μL kinase reaction buffer [50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1 mM ATP, 10 μCi [γ-32P] ATP (NEG502A001MC; Perkin-Elmer)] at 25°C for 1 h. Afterwards, the reactions were terminated by adding SDS loading dye, and samples were resolved by 10% SDS-PAGE. The phosphorylated bands indicated by 32P signal was visualized by autoradiography with a phosphor-plate on a Fujifilm FLA 3000 plus DAGE system.
Glanc M., Van Gelderen K., Hoermayer L., Tan S., Naramoto S., Zhang X., Domjan D., Včelařová L., Hauschild R., Johnson A., de Koning E., van Dop M., Rademacher E., Janson S., Wei X., Molnár G., Fendrych M., De Rybel B., Offringa R, & Friml J. (2021). AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells. Current Biology, 31(9), 1918-1930.e5.
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2

Recombinant GST-MEL1 and His-PID Protein Expression

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To express recombinant GST-MEL1 and His-PID proteins, the respective constructs were transformed into E. coli BL21 (DE3) cells for protein expression. Cultures at OD600 of 0.6 were induced with 0.5mM IPTG at 16°C overnight. Proteins were purified using Glutathione agarose for GST-MEL1 and Ni-NTA His binding resin for His-PID following the manufacturer’s instructions (Thermo Scientific). Purified proteins were analyzed by SDS-PAGE and visualized by Coomassie brilliant blue staining (Bio-Rad). In vitro protein kinase assay with [γ-32P] ATP was carried out as previously reported with minor modifications.42 (link) Recombinant GST-MEL1 (2 μg) and His-PID (5 μg) proteins were incubated together in 25 μL kinase reaction buffer [50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1 mM ATP, 10 μCi [γ-32P] ATP (NEG502A001MC; Perkin-Elmer)] at 25°C for 1 h. Afterward, the reactions were terminated by adding SDS loading dye, and samples were resolved by 10% SDS-PAGE. The phosphorylated bands indicated by 32P signal was visualized by autoradiography with a phosphor-plate on a Fujifilm FLA 3000 plus DAGE system.
Glanc M., Van Gelderen K., Hoermayer L., Tan S., Naramoto S., Zhang X., Domjan D., Včelařová L., Hauschild R., Johnson A., de Koning E., van Dop M., Rademacher E., Janson S., Wei X., Molnár G., Fendrych M., De Rybel B., Offringa R, & Friml J. (2021). AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells. Current Biology, 31(9), 1918-1930.e5.
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3

His-RCN1 Purification from E. coli

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A total of 35 ml lysate of His–RCN1 from 500 ml of E. coli culture was incubated with 1.5 ml Ni-NTA His binding resin (Thermo Scientific) for 30 min, and the supernatant was discarded. At the same time, 3 g Col-0 seedlings were homogenized into plant extraction buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% Tween-20, 1 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol) containing a protease inhibitor cocktail (cOmplete, Roche) and a protein phosphatase inhibitor tablet (PhosSTOP, Roche). The protein-bound resin was then incubated with protein extracts for 2 h at 4 °C. Afterwards, the column was washed twice with 10 ml wash buffer (1× TBS + 20 imidazole), and then eluted with 1 ml elution buffer (1× TBS + 250 imidazole) three times. Protein samples were checked by SDS–PAGE and visualized by Coomassie brilliant blue staining.
Roychoudhry S., Sageman-Furnas K., Wolverton C., Grones P., Tan S., Molnár G., De Angelis M., Goodman H.L., Capstaff N., Lloyd J.P., Mullen J., Hangarter R., Friml J, & Kepinski S. (2023). Antigravitropic PIN polarization maintains non-vertical growth in lateral roots. Nature Plants, 9(9), 1500-1513.
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4

Purification of Recombinant Proteins

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Recombinant proteins were expressed in the E. coli strain BL21 (DE3) with induction by 0.5 mM IPTG (Isopropyl β-D-1-Thiogalactopyranoside, 16°C, 12 h) and then purified using Ni-NTA His binding resin (Thermo Scientific) according to the manufacturer’s manual. The eluted samples were then purified with size exclusion chromatography, with a Superdex 200 increase column, on an ÄKTA pure chromatography system (GE Healthcare). Fractions were collected by 500 μL, and then analyzed by SDS-PAGE, followed by Coomassie brilliant blue (CBB, Bio-Safe Coomassie Stain #1610786 from BioRad) staining to check the protein quality.
Tan S., Abas M., Verstraeten I., Glanc M., Molnár G., Hajný J., Lasák P., Petřík I., Russinova E., Petrášek J., Novák O., Pospíšil J, & Friml J. (2020). Salicylic Acid Targets Protein Phosphatase 2A to Attenuate Growth in Plants. Current Biology, 30(3), 381-395.e8.
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5

Protein-Protein Interaction Assay

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GST—Cyld and GST protein were both individually expressed in BL21(DE3) E. coli. Cleared lysates were prepared and the soluble fusion proteins were purified on glutathione Sepharose 4B GST-tagged protein purification resin (GE Healthcare Life Sciences,) and tested by immunoblot using monoclonal anti-GST (Cell Signaling Technology). His-tagged NLRP6 expressed by the pET-30a(+) vector was transformed into BL21(DE3) E. coli and soluble fusion proteins were purified on Ni-NTA His-binding resin (Thermo Fisher Scientific). Purified GST—Cyld and GST were individually incubated with glutathione beads for approximately 4 h. Then the beads were collected by centrifugation, and washed five times with GST pulldown lysis buffer and TBS (1:1). Then, purified His—NLRP6 protein was added to glutathione beads and incubated at 4 °C overnight with gentle rocking. The beads were washed five times and resuspended in SDS—SDS—PAGE sample buffer. After boiling, the bound proteins were analyzed on 12% SDS—PAGE gel followed by immunoblotting using anti-His. His—NLRP6 protein was used as a control.
Mukherjee S., Kumar R., Lenou E.T., Basrur V., Kontoyiannis D.L., Ioakeimidis F., Mosialos G., Theiss A.L., Flavell R.A, & Venuprasad K. (2020). Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation. Nature immunology, 21(6), 626-635.
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6

Recombinant Protein Expression and Purification

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Coding sequences of RCN1, PID and PIN3HL were cloned into the pET28a vector (Novagen) by the restriction enzyme digestion method. Recombinant His-tagged proteins, including His–RCN1, His–PID and His–PIN3HL, were expressed in a BL21 (DE3) strain with induction by 0.5 mM isopropyl β-d-1-thiogalactopyranoside for 16 h at 12 °C. A total of 500 ml of E. coli culture was collected by centrifuge, resuspended in 35 ml 1× TBS (50 mM Tris–Cl and 150 mM NaCl; pH 7.6) buffer, and was then subjected to sonication. Proteins were then purified using Ni-NTA His binding resin (Thermo Scientific) following the manufacturer’s instruction. Eventually, the resultant protein samples were checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and visualized by Coomassie brilliant blue staining.
Roychoudhry S., Sageman-Furnas K., Wolverton C., Grones P., Tan S., Molnár G., De Angelis M., Goodman H.L., Capstaff N., Lloyd J.P., Mullen J., Hangarter R., Friml J, & Kepinski S. (2023). Antigravitropic PIN polarization maintains non-vertical growth in lateral roots. Nature Plants, 9(9), 1500-1513.
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7

Purification of Recombinant Proteins

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Recombinant proteins, including GST, GST-PDK1.1, GST-PDK1.2, GST-D6PKL1, His-D6PK, His-D6PK S345A , His-D6PK S345D , His-D6PKL2, His-PDK1.1N, His-PDK1.1C, His-PDK1.2N, His-PDK1.2C, His-PIN1HL, His-PIN2HL, and His-PIN3HL were expressed in an E. coli strain BL21 (DE3) with induction by 0.5 mM IPTG (Isopropyl β-D-1-Thiogalactopyranoside, 16°C, 12 h) and then purified using Ni-NTA His binding resin (Thermo Scientific) according to the manufacturer's manual. The eluted protein samples were further purified with size exclusion chromatography, with a Superdex 200 (10/30L) increase column (GE Healthcare), on an ÄKTA pure chromatography system. Protein samples were collected every 500 μL, and then checked by SDS-PAGE, visualized by the Coomassie brilliant blue staining (Bio-Safe™ Coomassie Stain #1610786, Bio-Rad). The protein concentration was qualified with the Bradford method (Quick Start™ Bradford Reagent, Bio-Rad).
Tan S., Zhang X., Kong W., Yang X.L., Molnár G., Vondráková Z., Filepová R., Petrášek J., Friml J, & Xue H.W. (2020). The lipid code-dependent phosphoswitch PDK1-D6PK activates PIN-mediated auxin efflux in Arabidopsis. Nature plants, 6(5).
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