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Ni nta agarose bead

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Ni-NTA agarose beads are a chromatography resin used for the purification of His-tagged proteins. The beads consist of a nickel-nitrilotriacetic acid (Ni-NTA) complex immobilized on an agarose matrix. These beads can selectively bind and capture proteins with a polyhistidine (His-tag) affinity tag, allowing for their efficient separation and purification from complex mixtures.

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Lab products found in correlation

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Ni-NTA agarose (1236 citations) Ni-NTA (356 citations) Ni-NTA beads (302 citations) NTA) agarose beads (11 citations) Ni-beads (11 citations) Ni-Agarose beads (6 citations) ) agarose beads (4 citations) Agarose (3 citations)

451 protocols using ni nta agarose bead

1

Progranulin Interactome Identification

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Serum-starved 5637 cells were stimulated with progranulin (40 nM) for 5 min and lysates collected in NP-40 lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (Thermo Scientifics, Waltham, MA USA). After preclearing for 1 h with Ni-NTA-agarose beads (QIAGEN, Valencia, CA, USA), lysates (10 mg) were first incubated with or without 7 μg of recombinant HIS-tagged progranulin overnight a 4°C and then with Ni-NTA-agarose beads for 1 h. Beads were washed with lysis buffer and resuspended in Laemmli buffer. Samples were separated by SDS-PAGE, visualized with Coomassie blue staining and processed for mass spectrometry at the Proteomic Core Facility of the Kimmel Cancer Center. Bands were excised and digested with trypsin after reduction and alkylation. Peptides were separated on a 10-cm C18 column and analyzed on a Thermo LCQ 3D ion trap using the nano-source. MS/MS spectra were searched on a local Mascot server against the Swissprot database. All peptides were identified with a confidence of at least 95%.
Xu S.Q., Buraschi S., Morcavallo A., Genua M., Shirao T., Peiper S.C., Gomella L.G., Birbe R., Belfiore A., Iozzo R.V, & Morrione A. (2015). A novel role for drebrin in regulating progranulin bioactivity in bladder cancer. Oncotarget, 6(13), 10825-10839.
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2

Bispecific Antibody Expression and Purification

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The bsAbs [HER2xCD3] and [(HER2)2xVγ9] as well as the control constructs [CD19xCD3] and [(HER2)2xCD89] were generated as published previously.12, 13 Briefly, Lenti‐X™ 293T cells were transfected with corresponding expression vectors coding for the bispecific single chain fragment variables (bsscFv) [HER2xCD3] and [CD19xCD3] or the tribodies [(HER2)2xVγ9] and [(HER2)2xCD89] using the calcium phosphate technique including 5 mM chloroquine. [HER2xCD3] and [CD19xCD3] molecules were purified by affinity chromatography using nickel‐nitrilotriacetic acid (Ni‐NTA) agarose beads (Qiagen, Hilden, Germany). Heterodimeric molecules [(HER2)2xVγ9] and [(HER2)2xCD89] composed of a light chain and a heavy chain derivative and tagged with a C‐terminal hexa‐histidine motif were purified from supernatant by two successive steps of affinity chromatography using CaptureSelect Fab kappa affinity matrix (BAC B.V., Naarden, Netherlands) and nickel‐nitrilotriacetic acid (Ni‐NTA) agarose beads (Qiagen) as described earlier.12, 13, 14 Purity and integrity of bsAbs were verified by capillary electrophoresis using an Experion automated electrophoresis system (Bio‐Rad, Kabelsketal, Germany) and size exclusion chromatography.
Oberg H., Janitschke L., Sulaj V., Weimer J., Gonnermann D., Hedemann N., Arnold N., Kabelitz D., Peipp M., Bauerschlag D, & Wesch D. (2019). Bispecific antibodies enhance tumor‐infiltrating T cell cytotoxicity against autologous HER‐2‐expressing high‐grade ovarian tumors. Journal of Leukocyte Biology, 107(6), 1081-1095.
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3

Purification of Recombinant Endophilin A1 in E. coli

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Recombinant proteins were expressed as N-terminal his6-SUMO-tagged fusion proteins in the BL21(DE3) E. coli strain. Protein purification and tag removal were carried out using published protocols (Poudel et al., 2016 (link)). Briefly, the bacteria culture was grown in Luria Broth medium at 37°C. When OD600 reached 1.0, isopropyl-D-thiogalactopyranoside was added to the bacterial culture (0.2mM) to induce protein expression. The bacterial culture was further grown overnight at 15°C. Bacteria harvested by centrifugation was lysed by a microfluidizer in the lysis buffer (20 mM HEPES, pH 8.0, 300 mM NaCl, 15 mM imidazole). Proteins were purified using NiNTA-agarose beads (Qiagen, Valencia, CA), and then eluted with a lysis buffer plus 250 mM imidazole. Recombinant endophilinA1 variants were expressed as his6-SUMO-tagged fusion proteins. The SUMO protease ULP1 (ubiquitin-like-specific protease 1) was used to cleave off his6-SUMO tags, resulting in endophilin A1 variants with native N-terminal amino acid compositions. His6-SUMO fragments cleaved off by ULP1 were removed using NiNTA-agarose beads (Qiagen, Valencia, CA). Purified proteins were dialyzed against the HEPES buffer (50 mM HEPES, pH 7.4, 150mM NaCl) plus 1 mM DTT. Proteins were stored at 4°C.
Zhang L., Wang Y., Dong Y., Pant A., Liu Y., Masserman L., Xu Y., McLaughlin RN J.r, & Bai J. (2022). The endophilin curvature-sensitive motif requires electrostatic guidance to recycle synaptic vesicles in vivo. Developmental cell, 57(6), 750-766.e5.
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4

IpaH Ubiquitination Assay Protocol

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Shigella flexneri genes encoding IpaH proteins were cloned into pGEX4T-1, expressed in E. coli BL21 and purified using glutathione coated beads. His6-UbcH5b and His6-UbE1 were expressed in E. coli BL21 and purified using Ni-NTA agarose beads (Qiagen). LUBAC (Flag-HOIL-1L/Myc-HOIP/Flag-His6-SHARPIN) was expressed in HeLa cells and purified using Ni-NTA agarose beads (Qiagen). LUBAC purified from HeLa cells was then used in an Ub reaction, containing 0.1 μM UbE1 (E1), 2.5 μM UbcH5b (E2), 5 μM (Flag, HA or His6 tagged) Ub (BostonBiochem), 0.5 mM MgSO4, 0.5 mM ATP, 20 units/ml inorganic pyrophosphatase (Sigma Aldrich), 1 mM DTT and 50 nM GST-IpaH (E3). After 0, 20, 60 or 90 minutes incubation at 37°C samples were electrophoresed on an SDS PAGE buffer and a Western blot was stained with anti-Myc antibodies. Wild type Ub (Flag, HA, His6 tagged), K48 only Ub (His6 tagged) and K48R (His6 tagged) were purchased from BostonBiochem.
de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.
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5

Recombinant PRPS2 Protein Purification

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Full-length wild-type or mutant human PRPS2 sequences with a C-terminal 6 × His-tag were cloned into a modified pRSFDuet vector and expressed in E. coli Transetta (DE3) cells. After induction with 0.1 mM IPTG at the OD600 range of 0.5 ~ 0.8, the cells were cultured at 37 °C for 4 h and pelleted by centrifugation at 4,000 r.p.m. for 10 min. The harvested cells were sonicated under the ice in lysis buffer (50 mM Tris–HCl pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 1 mM PMSF, 5 mM β-mercaptoethanol, 5 mM benzamidine, 2 μg/ml leupeptin and 2 μg/ml pepstatin). After ultrasonication, the cell lysate was then centrifuged (15,000 r.p.m.) at 4 °C for 45 min. The supernatant was collected and incubated with equilibrated Ni–NTA agarose beads (Qiagen) for 1 h. and purified by Ni–NTA agarose beads (Qiagen). Lysis buffer with 50 mM imidazole was used to wash the column. And target proteins were eluted with lysis buffer with 250 mM imidazole. Further purification was performed in column buffer (25 mM Tris HCl pH 8.0 and 150 mM NaCl) using HiLoad Superdex 200 gel-filtration chromatography (GE Healthcare). The peak fractions were collected, concentrated, and stored in small aliquots at − 80 °C. All the experiments were performed at 4 °C.
Lu G.M., Hu H.H., Chang C.C., Zhong J., Zhou X., Guo C.J., Zhang T., Li Y.L., Yin B, & Liu J.L. (2023). Structural basis of human PRPS2 filaments. Cell & Bioscience, 13, 100.
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6

IpaH Ubiquitination Assay Protocol

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Shigella flexneri genes encoding IpaH proteins were cloned into pGEX4T-1, expressed in E. coli BL21 and purified using glutathione coated beads. His6-UbcH5b and His6-UbE1 were expressed in E. coli BL21 and purified using Ni-NTA agarose beads (Qiagen). LUBAC (Flag-HOIL-1L/Myc-HOIP/Flag-His6-SHARPIN) was expressed in HeLa cells and purified using Ni-NTA agarose beads (Qiagen). LUBAC purified from HeLa cells was then used in an Ub reaction, containing 0.1 μM UbE1 (E1), 2.5 μM UbcH5b (E2), 5 μM (Flag, HA or His6 tagged) Ub (BostonBiochem), 0.5 mM MgSO4, 0.5 mM ATP, 20 units/ml inorganic pyrophosphatase (Sigma Aldrich), 1 mM DTT and 50 nM GST-IpaH (E3). After 0, 20, 60 or 90 minutes incubation at 37°C samples were electrophoresed on an SDS PAGE buffer and a Western blot was stained with anti-Myc antibodies. Wild type Ub (Flag, HA, His6 tagged), K48 only Ub (His6 tagged) and K48R (His6 tagged) were purchased from BostonBiochem.
de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.
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7

TRPP3 N-C Terminal Interaction

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Purified GST-tagged human TRPP3 N terminus (M1-L95; 2 μg) from E. coli was incubated with the same amount of purified His-tagged human TRPP3 C-terminal fragment (I560-K660) from E. coli in the CelLytic-M lysis buffer (Sigma). The mixture was incubated at RT for 1 hr with gentle shaking, followed by another hr of incubation after addition of 10 μL 50% Ni-NTA agarose bead (QIAGEN, Hilden, Germany). The beads were then washed three times with PBS buffer supplemented with 1% Nonidet P-40, and the remaining proteins were eluted using SDS loading buffer and resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was then immunoblotted with His and GST antibodies.
Zheng W., Cai R., Hofmann L., Nesin V., Hu Q., Long W., Fatehi M., Liu X., Hussein S., Kong T., Li J., Light P.E., Tang J., Flockerzi V., Tsiokas L, & Chen X.Z. (2018). Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2. Cell reports, 22(6), 1560-1573.
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8

TRPP3 N-C Terminal Interaction

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Purified GST-tagged human TRPP3 N terminus (M1-L95; 2 μg) from E. coli was incubated with the same amount of purified His-tagged human TRPP3 C-terminal fragment (I560-K660) from E. coli in the CelLytic-M lysis buffer (Sigma). The mixture was incubated at RT for 1 hr with gentle shaking, followed by another hr of incubation after addition of 10 μL 50% Ni-NTA agarose bead (QIAGEN, Hilden, Germany). The beads were then washed three times with PBS buffer supplemented with 1% Nonidet P-40, and the remaining proteins were eluted using SDS loading buffer and resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was then immunoblotted with His and GST antibodies.
Zheng W., Cai R., Hofmann L., Nesin V., Hu Q., Long W., Fatehi M., Liu X., Hussein S., Kong T., Li J., Light P.E., Tang J., Flockerzi V., Tsiokas L, & Chen X.Z. (2018). Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2. Cell reports, 22(6), 1560-1573.
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9

Assessing Ubiquitination via Affinity Purification

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To assess ubiquitination, the pCMV‐HA‐TβRI, pCMV‐His‐Ub and pCMV5B‐Flag‐SMURF2‐WT expression vectors were co‐transfected with mimics or inhibitors of miR‐NC, miR‐195 and miR‐497 using the calcium chloride transfection method. The pCMV‐HA‐TβRI was a kind gift from K.‐M. Yang (Seoul National University) and pCMV5B‐Flag‐SMURF2‐WT was a gift from J. Wrana (Addgene plasmid # 11746) (Kavsak et al., 2000). The cells were treated with 10 μm MG132 prior to harvesting to inhibit proteasomal degradation.
Transfected HEK293T cells were harvested with urea lysis buffer (8 m urea, 10 mm imidazole, 300 mm NaCl, 50 mm Na2HPO4, 50 mm Tris and 1 mm PMSF, pH 8.0). Cell lysates were lysed by sonication and mixed with Ni‐NTA agarose bead (Qiagen) for 4 h at 4 °C in a rotator. The agarose was washed five times with urea lysis buffer (8 m urea, 20 mm imidazole, 300 mm NaCl, 50 mm Na2HPO4, 50 mm Tris and 1 mm PMSF, pH 8.0). The bound protein was eluted off the Ni‐NTA agarose bead in 2× SDS sample buffer by boiling at 95 °C. Affinity purified protein samples were analyzed by SDS/PAGE.
Chae D., Park J., Cho M., Ban E., Jang M., Yoo Y.S., Kim E.E., Baik J, & Song E.J. (2019). MiR‐195 and miR‐497 suppress tumorigenesis in lung cancer by inhibiting SMURF2‐induced TGF‐β receptor I ubiquitination. Molecular Oncology, 13(12), 2663-2678.
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10

Purification of Nup Protein Complex

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The structured region of central channel complex from Rattus norvegicus was cloned in modified pET28a as described earlier.46 The construct contains thrombin cleavage site at the N terminus of His6 tagged Nup58 (239–415) followed by Nup62 (322–525) and Nup54 (332–510). The conserved region of Nup58 in Chaetomium thermophilum Nup49 (246–470) was gene synthesized (Invitrogen) and replaced on the above construct. Both the constructs were subjected to the same protocol for Ni‐NTA affinity purification.46 Briefly, BL21(DE3)‐RIL strain of Escherichia coli was transformed with the construct and the culture(2 L) was induced with 0.5 mM IPTG at OD ~0.6 followed by incubation of 8 h at 18°C and purified using Ni‐NTA agarose bead (Qiagen). The purified protein complex was dialysed against buffer (Tris–HCl pH 8, 250 mM NaCl, 1 mM DTT) and digested with thrombin at 4°C. The digested protein was concentrated using a 3 kDa cutoff concentrator (Merck) and subjected for SEC using superdex 200, 10/300 GL column (GE Healthcare) in SEC buffer (Tris–HCl pH 8, 250 mM NaCl, 0.5 mM EDTA, 1 mM DTT) at 4°C.
Chopra K., Bawaria S, & Chauhan R. (2018). Evolutionary divergence of the nuclear pore complex from fungi to metazoans. Protein Science : A Publication of the Protein Society, 28(3), 571-586.
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