Affinity precipitation assays using Ni-NTA beads (GE Healthcare) were performed with His-S RBD and FITC-SAPs using previously described methods.44 (link) Ni-NTA beads were equilibrated in binding buffer (50 mM Tris pH 7.5, 250 mM NaCl, 50 mM imidazole, and 2 mM β-mercaptoethanol). Binding reactions were setup by incubating equilibrated Ni-NTA beads with His-S RBD (1 μM) and increasing concentrations (3, 1, 0.5, 0.25, and 0.125 mM) of indicated FITC-SAP in the binding buffer and incubated for 1 h at 4 °C. In parallel, control reactions with 3 mM of indicated FITC-SAP without His-S RBD were preformed to rule out nonspecific FITC-SAP binding to the Ni-NTA beads. Reactions were spun and washed three times in the binding buffer to remove unbound proteins/peptides. The resulting protein–peptide complexes bound to the beads were extracted using NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen), subjected to SDS-PAGE analysis, and visualized by Coomassie staining or fluorescence detection at 488 nm. Resulting FITC-labeled bands were quantified using ImageJ software. To estimate Kd values for FITC-SAP binding to His-S RBD, the data were fit to the Hill equation using Origin 8 software (OriginLab).
Ni nta beads
Manufactured by GE Healthcare
Sourced in China
Ni-NTA beads are a type of affinity chromatography resin used for the purification of recombinant proteins. The beads are coated with nickel ions (Ni2+) that bind to histidine-tagged proteins, allowing for their selective capture and subsequent elution. This technology enables efficient and selective purification of target proteins from complex biological samples.
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Lab products found in correlation
Tripletof 5600 mass spectrometer, by AB Sciex (1 mentions)
Scaffold software, by Proteome Software (1 mentions)
Mascot, by Matrix Science (1 mentions)
Protein a g beads, by Beyotime (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Dna marker, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Kod plus dna polymerase, by Toyobo (1 mentions)
Instantblue coomassie stain, by Abcam (1 mentions)
Slide a lyzer, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Abcam (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Cation exchange column, by GE Healthcare (1 mentions)
Superdex 200 gel filtration column, by GE Healthcare (1 mentions)
Superdex 200 increase, by GE Healthcare (1 mentions)
Resource s column, by GE Healthcare (1 mentions)
Pgex 6p 1 expression vector, by GE Healthcare (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Transetta de3 chemically competent cell, by Transgene (1 mentions)
27 protocols using ni nta beads
1
Affinity Precipitation of SARS-CoV-2 RBD
2
Immunoprecipitation and Mass Spectrometry Analysis
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HEK 293T cells were lysed and centrifuged. The supernatants were incubated with primary antibody at 4 °C overnight and then captured with protein A+G beads (Beyotime, China) or Ni-NTA beads (GE, USA). The proteins labeled with HA were enriched by immunoprecipitation, and the reaction products were subjected to SDS-PAGE electrophoresis and coomassie blue staining. Different bands in the experimental groups were excised, and in-gel tryptic digested. The digested peptide was analyzed as previously described42 (link) by LC–MS/MS on a Triple TOF 5600 mass spectrometer (AB Sciex, Framingham, MA, USA). MS/MS data were searched against the SwissProt human database (20227 entries) using Mascot (version 2.4.01; Matrix Science, London, UK), and processed using Scaffold software (version 4.0.7; Proteome Software Inc., Portland, OR, USA).
3
Purification of GST and His6-tagged Proteins
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GST or His6-tag protein plasmids were expressed in the BL21(DE3) or ROSETTA(DE3) strains and grown firstly at 37 ˚C in LB medium supplemented with appropriate antibiotics. When OD600 reached 0.6–0.8, protein expression was then induced at 16 ˚C with 0.5 mM IPTG for more than 16 h. To purify His6-tagged proteins, bacteria were harvested and lysed in Ni-NTA Binding buffer (50 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, 0.1% TritonX-100, 1 mM DTT). Proteins were purified with affinity chromatography using Ni-NTA beads (GE) according to the manufacturer’s instruction with wash buffer (50 mM Na2HPO4, 500 mM NaCl, 40 mM imidazole, 0.1% TritonX-100, 1 mM DTT) and elution buffer (50 mM Na2HPO4, 300 mM NaCl, 250 mM imidazole). For GST-fusion proteins, bacteria were lysed in NETN buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT). Purifications were performed by affinity chromatography using Glutathione Sepharose Fast Flow beads (Sangon Biotech, China). Beads were washed with NETN buffer and then eluted by elution buffer (200 mM Tris-Cl pH 8.0, 20 mM Glutathione). All eluted proteins were further dialyzed in PBS overnight at 4 ˚C. Recombinant proteins were concentrated and then stored at −80 ˚C. Protein concentrations were determined using Bradford colorimetric assays, with their protein purities examined with SDS-PAGE followed by Coomassie Blue staining.
4
Purification of Sec23/24 and WFS1 Proteins
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E. coli Rosetta BL21 (DE3) cells expressing SumoHis-tagged Sec23/24 or the GST-tagged N-terminus of WFS1 were cultured with Terrific Broth (TB) medium at 37 °C and induced by 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 16–18 h. When the OD600 reached 2.0, the cells were harvested and sonicated in PBS buffer with 400 mM NaCl and 0.01% Triton X-100, and then centrifuged at 32,000g for 30 min at 4 °C. The supernatants were incubated with Ni-NTA beads (GE Healthcare, Piscataway, NJ, USA) or 4B GST beads (GE Healthcare, Piscataway, NJ, USA) for SumoHis-tagged proteins or GST-tagged proteins for 1 h, respectively. Then, washed with PBS for six times and eluted by PBS with 300 mM Imidazole. The eluted proteins were dialyzed against buffer B (20 mM Tris-HCl pH 8.0, 300 mM NaCl, and 0.5 mM EDTA) and stored at −80 °C for the subsequent binding assay.
5
Recombinant Expression of TBC1D15-GAP Proteins
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The E. coli strains TG1 and BL21 (DE3); the plasmids pETduet-His-sumo-Shark-TBC1D15-GAP, pETduet-His-sumo-Sus-TBC1D15-GAP, and pETduet-His-sumo-Homo-TBC1D15-GAP; and the vectors pETduet-His-sumo-Rab4/Rab5/Rab7/Rab11 were gifted from Professor Jianhong Shu of Zhejiang Sci-Tech University. A Plasmid Mini Kit, Agarose Gel Extraction Kit and PCR Clean Kit were purchased from Axyen (Beijing, China). Restriction enzymes, T4 DNA ligase and DNA markers were obtained from Takara (Beijing, China). KOD-plus DNA Polymerase was purchased from TOYOBO (Shanghai, China). Protein marker was obtained from Thermo Fisher Scientific (Shanghai, China). GTP powder was obtained from Aladdin (Shanghai, China). Ni–NTA beads and 50 ml gravity columns were obtained from GE Healthcare (Beijing, China).
6
Production and Purification of Protein Immunogens
7
Purification of Recombinant FHL-1 Proteins
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Conditioned media from each of the 20 dishes (17.5 ml/dish) were collected and pooled after 24, 48, 72, and 144 h. Total conditioned media after 144 h (1,400 mls total) was diluted with the addition of 600 mls 50 mM HEPES, 500 mM NaCl, 20 mM Imidazole, pH 7.5. To this, 12 ml of NiNTA resin (Expedeon) was added and incubated overnight with rotation at 4 °C. The NiNTA beads were collected by passing the media through empty PD10 columns with a filter (GE Healthcare) by gravity flow (500 ml media per PD10 column, i.e. four PD10 columns in total). The beads were then washed with 10 ml of wash buffer (50 mM HEPES, 500 mM NaCl, 20 mM Imidazole, pH 7.5). Finally, wild-type FHL-1 or RGD-null FHL-1 protein was eluted using 16 ml of elution buffer (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.5). Eluted protein was dialysed back into wash buffer overnight at 4 °C before being concentrated further by addition to 1.5 ml NiNTA beads and eluted in 6 × 1 ml aliquots. Purified protein aliquots were dialysed into 20 mM glycine, 125 mM NaCl, pH 9.0 using a dialysis cassette Slide-A-Lyzer (Fisher, cat no 10759784) with a 10 kDa cut off. The purified recombinant proteins (i.e. FHL-1 and RGD-null FHL-1) were assessed for purity by SDS-PAGE, and visualised by staining the gels for 60 min at room temperature with Instant Blue Coomassie stain (Expedeon, Cambridge, UK).
8
Purification of Mutant CSF-1R Receptor
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Wild-type CSF-1R (residues 542–919, △696–741, C677T, C830S, C907T) and CSF-1RT663I carrying an N-terminal 6×His tag was cloned into a pFastBac expression vector, and was expressed in SF9 cells using the Invitrogen Bac-to-Bac Baculovirus Expression System41 (link). For crystallization, CSF-1R was co-expressed with N-terminal GST-tagged YOPH to induce non-phosphorylated proteins. The harvested cell pellets were lysed by sonication in lysis buffer consisting of 40 mM K-phosphate, pH 8.0, 200 mM NaCl, 0.5 mM PMSF, and protease inhibitor mixture (EDTA-free, Roche). After centrifugation, the clarified cell lysate was supplemented with 20 mM imidazole, and incubated with Ni-NTA beads (GE Healthcare). The Ni-NTA bead-bound CSF-1R protein was eluted using 25 mM HEPES, pH 7.0, 150 mM NaCl and 200 mM imidazole, and was subsequently supplemented with 10 mM DTT after elution. CSF-1R was further purified using a cation exchange column (GE Healthcare) followed by a Superdex200 gel filtration column (GE Healthcare) equilibrated in 20 mM HEPES, pH 7.0, 150 mM NaCl and 10 mM DTT.
9
Purification of Bacterial and Insect-Expressed Proteins
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Both bacterial expressed and insect cell-expressed proteins were purified using Ni-affinity chromatography and size exclusion chromatography (Superdex 200 10/300 GL). The bacteria cells were lysed on ice by sonication in Buffer A (50 mM Tris-HCl pH 8, 300 mM NaCl and 15 mM imidazole), added with 5 mM β-Mercaptoethanol if purifying proteins under a reducing condition. The supernatant that was separated from the E. coli lysate through centrifugation was incubated with Ni-NTA beads (GE Healthcare) for one hour at 4 °C with rotation. The insect cell medium that contained the expressed secreted protein was loaded onto a pre-packed Ni column directly after pH adjustment. The Ni-NTA beads bound with the protein were washed 3 times with Buffer B (50 mM Tris-HCl at pH 8, 300 mM NaCl, 35 mM imidazole, with or without 5 mM β-mercaptoethanol). The proteins were eluted with Buffer C (50 mM Tris-HCl at pH 8, 300 mM NaCl, 150 mM imidazole, with or without 5 mM β-mercaptoethanol). Subsequently, the concentrated protein eluent was subjected to size exclusion chromatography using Buffer D (20 mM Tris-HCl at pH 8 and 150 mM NaCl, with or without 2.5 mM DTT).
10
Purification of Mouse Cortactin Fragments
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Three fragments of mouse cortactin (Uniprot: Q60598) comprising residues Gly83-Phe324 or Gly83-Thr401 of cortactin were subcloned into the pGEX6p-1 expression vector (GE), with an N-terminal glutathione S-transferase (GST) affinity tag followed by a PreScission protease site. These were transformed into Escherichia coli strain Rosetta(DE3) (Novagen) for expression. Production of the targeted proteins was induced by 0.2 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) at 16 °C overnight. Cells were harvested and lysed in 1x PBS buffer supplemented with protease inhibitors (Roche) and clarified supernatant was loaded onto glutathione-Separose 4B beads (GE) or Ni-NTA beads (GE). GST-cortactin was then digested with PreScission protease on-column overnight at 4 °C. The cleaved target protein was applied to a Resource S column (GE) in buffer of 20 mM MES pH 6, 5% glycerol, 1 mM DTT, and eluted with an NaCl gradient from 10 mM to 500 mM. The elution peak was loaded onto a Superdex 200 increase (GE) column. Each construct resulted in a single peak of cortactin protein. The final purified fragments of cortactin each contain N-terminal vector derived residues GPLGS followed by cortactin. The constructs are termed cortactinCR (residues Gly83-Phe324) and cortactinCRH (Gly83-Thr401).
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