Approximately 100 uL of packed Ni beads (Ni-NTA Superflow beads, Qiagen) were washed three times in water and stripped with 100 mM EDTA pH ~8.9 for 30 min. Beads were then washed three times with water and once with 80% ACN in 0.1% trifluoroacetic acid. Lyophilized iTRAQ samples were resuspended in 1.5 mL of ACN in 0.2% TFA and incubated with prepared beads for 1 hour at RT. Beads were then washed three times with 80% ACN in 0.1% TFA, and phosphopeptides were eluted from beads with 2 incubations in 75 uL of 1.4% Ammonia. Samples were then vacuum centrifuged down to ~20 uL. 2 uL of 200 mM ammonium formate pH 10 was added and samples were directly analyzed by mass spectrometry.
Ni nta superflow beads
Manufactured by Qiagen
Sourced in Germany, United States
Ni-NTA Superflow beads are a type of agarose-based chromatography resin designed for the purification of histidine-tagged proteins. These beads contain nickel-nitrilotriacetic acid (Ni-NTA) as the ligand, which binds to the histidine tag on the target protein, allowing for efficient capture and purification. The Superflow variant offers improved flow characteristics compared to standard Ni-NTA resins, making the purification process more efficient.
Automatically generated - may contain errors
Lab products found in correlation
Vivaspin columns, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Mms 156p, by Fortrea (2 mentions)
Coomassie brilliant blue, by Carl Roth (2 mentions)
Champion pet sumo vector, by Thermo Fisher Scientific (2 mentions)
Edta free protease inhibitor cocktail, by Merck Group (2 mentions)
Hitrap heparin hp column, by GE Healthcare (2 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
E coli rosetta 2 de3 singles competent cells, by Merck Group (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (1 mentions)
Pfastbac nt topo vector, by Thermo Fisher Scientific (1 mentions)
Asolectin, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Msp1e3d1, by Addgene (1 mentions)
Leupeptin, by MP Biomedicals (1 mentions)
Ab18738, by Abcam (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Maxis 4g, by Bruker (1 mentions)
34 protocols using ni nta superflow beads
1
Phosphopeptide Enrichment and Analysis
2
Recombinant ALK Tyrosine Kinase Domain Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA encoding ALK residues 1090–1416, which includes whole tyrosine kinase domain (TKD), was amplified with Phusion High-Fidelity DNA polymerase (ThermoScientific, Waltham, MA, USA) and cloned into pFastBac/NT-TOPO vector (Invitrogen, Carlsbad, CA, USA) for expression of 6x histidine-tagged recombinant ALK TKD proteins in Sf21 cells. Recombinant baculovirus was generated using the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA, USA). Sf21 cells were infected with P2 virus stock for three days at 27 °C, and then lysed in native Ni-NTA lysis buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, and protease inhibitor cocktail). His-tagged protein was recovered from the lysis supernatant using Ni-NTA Superflow beads (Qiagen, Hilden, Germany). After 2× wash, the resins were incubated in 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 10 mM MgCl2 and 2 mM ATP at 30 °C for 1 h to achieve further ALK TKD autophosphorylation. After 2× additional washes, ALK TKD proteins were eluted and their concentrations were determined by absorbance at 280 nm. Purity of ALK TKD proteins was assessed by SDS-PAGE and Coomassie blue staining.
3
Purification of Drosophila Pumilio Protein Domains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Drosophila Pumilio domains (PUM-D1, PUM-D3, Pum-D1D3) fused to V5-6xHis were subcloned into pET 28a and pET26a vectors for protein expression in bacteria. Plasmids were transformed into BL21(DE3) competent cells and recombinant proteins were induced with 1 mM IPTG at 25 °C. Bacteria were lysed in lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH8.0, 0,1% Triton, Protease inhibitors). Samples were lysed by sonication, centrifuged twice at 14,000 × g for 15 min, and the cleared supernatant was bound to Ni-NTA Superflow beads (Qiagen) by gravity filtration. Unbound proteins were washed with lysis buffer at pH 8.0 supplemented with increasing amounts of imidazole (5 mM, 40 mM, 60 mM). Recombinant proteins were eluted from beads in lysis buffer with 100–250 mM imidazole at pH 8.0. For recombinant proteins retained in inclusion bodies (PUM-D3), solubilization was done by including 6M urea in lysis buffer. All purified Pumilio recombinant proteins were dialized overnigth at 4 °C, against a final buffer (20 mM Hepes, 200 mM NaCl, 5% Glicerol, 2 mM DTT) and concentrated with appropriate MW cut-off Vivaspin columns (Merck). The concentration of purified proteins was determined by colorimetric assay (Bio-Rad DC Protein Assay) and verified by electrophoresis alongside of BSA standards with Coomassie staining.
4
Purification and Reconstitution of Membrane Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Penicillin and streptomycin were from Gibco-BRL (Breda, The Netherlands). Leupeptin was obtained from MP Biomedicals (Santa Ana, CA, USA), heptakis (2,6-di-O-methyl)-β-cyclodextrin (β-cyclodextrin) and asolectin from Sigma-Aldrich Co. (St. Louis, MO, USA). Grace's and Insect-Xpress insect media were from Lonza (Breda, The Netherlands); 11-cis retinal was a generous gift from Dr. Rosalie Crouch (Medical University of South Carolina, Charleston, SC, USA) through financial support from NEI; DDM and n-nonyl-β-glucoside (NG) were obtained from Serva Electrophoresis GmbH (Heidelberg, Germany). The plasmid for ZAP1 (Banerjee et al., 2008) was provided by Dr. Thomas Sakmar (The Rockefeller University, New York, NY, USA). The plasmids for MSP1D1 and MSP1E3D1 ( Bayburt et al., 2002) were acquired through Addgene (Cambridge, MA, USA). Ni-NTA Superflow beads were purchased from Qiagen Benelux B.V. (Venlo, The Netherlands) and the high affinity analog of the Gtα C-terminal peptide (NH 2 -VLEDLKSCGLF-COOH) from United Peptide Corporation (Chantilly, VA, USA).
Buffer A: 40 mm Tris, 0.3 m NaCl, and 1 mm NaN3 (pH 8.0). Buffer B: 20 mm Bis/Tris propane, 5 mm MgCl 2 , 1 mm dithioerythritol (DTE), 1 mm benzamidine, and 5 μm Leupeptin (pH 7.6). Buffer C: 20 mm Bis/ Tris propane, 1 m NaCl, 1 mm histidine, and 5 μm Leupeptin (pH 7.2). Buffer D: buffer B+100 mm NG.
Buffer A: 40 mm Tris, 0.3 m NaCl, and 1 mm NaN3 (pH 8.0). Buffer B: 20 mm Bis/Tris propane, 5 mm MgCl 2 , 1 mm dithioerythritol (DTE), 1 mm benzamidine, and 5 μm Leupeptin (pH 7.6). Buffer C: 20 mm Bis/ Tris propane, 1 m NaCl, 1 mm histidine, and 5 μm Leupeptin (pH 7.2). Buffer D: buffer B+100 mm NG.
5
Purification and Cleavage of His-Tagged Bacterial Proteins
6
Affinity Purification of Cdc14 Phosphatase
7
Affinity Purification of Cdc14 Phosphatase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10 μl of Ni-NTA Superflow beads (Qiagen) were equilibrated in 20 mM Tris/HCl pH 7.5, 200 mM NaCl, 0.2% NP-40, 20 mM imidazole, following which they were chelated to 30 pmol His6-Net11-600 or His6-GFP for one hour at 4 °C. 30 pmol GST-Cdc14, GST-Cdc14 TAB6 or GST-Cdc14 W108A were added to the beads for 15 minutes at 30 °C at a final concentration of 0.5 μM. Following washes in the same buffer, the beads were boiled in SDS-PAGE loading buffer and eluates analyzed by SDS-PAGE followed by Coomassie Blue staining.
8
Protein-Protein Interaction Assay using Ni-NTA Beads
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This method was adapted from (Egan et al., 2015 (link)). Proteins (1 μM) were mixed in 200 μl binding buffer (10 mM Hepes/NaOH, 10 mM MgCl2, 150 mM NaCl, 0.05% Triton X-100, pH 7.5). Samples were applied to 100 μl of washed and equilibrated Ni-NTA superflow beads (Qiagen, The Netherlands) and incubated overnight at 4˚C with gentle mixing. The beads were then washed with wash buffer (10 mM Hepes/NaOH, 10 mM MgCl2, 500 mM NaCl, 50 mM imidazole, 0.05% Triton X-100, pH 7.5) and boiled in SDS–PAGE loading buffer. Beads were pelleted by centrifugation and samples analysed by SDS–PAGE. Gels were stained with Coomassie brilliant blue (Roth, Germany).
9
Nickel Pull-Down Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nickel pull‐down assays were performed in buffer A plus 300 mM NaCl and 10 mM imidazole pH 8.0. Then, 10 μg bait (His6‐MBP‐N3) was mixed with equal weights of each prey protein in 50 μl total reaction volume and incubated on ice for 30 min (5 μl “load” sample removed). Total volume of 30 μl of a 50% slurry of Ni‐NTA Superflow beads (Qiagen) was added and the mixture was incubated with occasional mixing on ice for 30 min. Beads were washed three times with 1 ml buffer, then bound proteins were eluted with the addition of 30 μl buffer A plus 300 mM NaCl and 250 mM imidazole pH 8.0. Then, 10 μl of each eluate was analyzed by SDS‐PAGE alongside the load samples.
10
Purification of Recombinant Pumilio Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cairrão et al, 10
proteins were induced with 1mM IPTG at 25ºC. Bacteria were lysed in lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH8.0, 0,1% Triton, Protease inhibitors). Samples were lysed by sonication, centrifuged twice at 12,000rpm for 15min, and the cleared supernatant was bound to Ni-NTA Superflow beads (Qiagen) by gravity filtration. Unbound proteins were washed with lysis buffer at pH 8.0 supplemented with increasing amounts of imidazole (5mM, 40mM, 60mM). Recombinant proteins were eluted from beads in lysis buffer with 100-250 mM imidazole at pH 8.0. For recombinant proteins retained in inclusion bodies (PUM-D3), solubilization was done by including 6M urea in lysis buffer. All purified Pumilio recombinant proteins were dialized overnigth at 4°C, against a final buffer (20mM Hepes, 200 mM NaCl, 5% Glicerol, 2mM DTT) and concentrated with appropriate MW cut-off Vivaspin columns (Merck). The concentration of purified proteins was determined by colorimetric assay (Bio-Rad DC Protein Assay) and verified by electrophoresis alongside of BSA standards with Coomassie staining.
proteins were induced with 1mM IPTG at 25ºC. Bacteria were lysed in lysis buffer (500 mM NaCl, 50 mM Tris-Cl, pH8.0, 0,1% Triton, Protease inhibitors). Samples were lysed by sonication, centrifuged twice at 12,000rpm for 15min, and the cleared supernatant was bound to Ni-NTA Superflow beads (Qiagen) by gravity filtration. Unbound proteins were washed with lysis buffer at pH 8.0 supplemented with increasing amounts of imidazole (5mM, 40mM, 60mM). Recombinant proteins were eluted from beads in lysis buffer with 100-250 mM imidazole at pH 8.0. For recombinant proteins retained in inclusion bodies (PUM-D3), solubilization was done by including 6M urea in lysis buffer. All purified Pumilio recombinant proteins were dialized overnigth at 4°C, against a final buffer (20mM Hepes, 200 mM NaCl, 5% Glicerol, 2mM DTT) and concentrated with appropriate MW cut-off Vivaspin columns (Merck). The concentration of purified proteins was determined by colorimetric assay (Bio-Rad DC Protein Assay) and verified by electrophoresis alongside of BSA standards with Coomassie staining.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!