The rWLP contains a His6 tag in its sequence, and Ni-NTA beads (Sangon, China) were used for binding the rWLP. After binding with rWLP, the Ni-NTA beads were washed with binding buffer (20 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH 8.0) and then incubated with total proteins extracted from the shell of M. coruscus [8 (link)] for 4 h at 4°C and then washed with elution buffer (20 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole, pH 8.0). The eluted protein samples were analysed by LC-MS/MS after digestion with trypsin. The LC-MS/MS experiments were performed on a Q Exactive Plus MS coupled with Easy-nLC (Thermo Scientific). The MS data were analysed using MaxQuant software (version 1.6.1.0.), and searched against the mantle transcriptome database of M. coruscus (Accession: SRX792025) [8 (link)]. The database search results were filtered and exported with a <1% false discovery rate (FDR) at the peptide-spectrum-matched level and protein level.
Ni nta beads
Manufactured by Sangon
Sourced in China
Ni-NTA beads are a type of affinity chromatography resin used for the purification of His-tagged proteins. They consist of nickel-nitrilotriacetic acid (Ni-NTA) covalently attached to agarose beads. The nickel ions on the beads bind to the histidine residues on the target protein, allowing it to be captured and separated from other components in the sample.
Automatically generated - may contain errors
4 protocols using ni nta beads
1
Identification of Proteins Interacting with Recombinant Shell Protein
2
Affinity Purification of rVDCP Interactors
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The rVDCP protein contains a His 6 tag in its sequence, and Ni-NTA beads (Sangon, China) were used to bind the tagged rVDCP protein.
After binding to rVDCP, the Ni-NTA beads were washed with binding buffer (20 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH 8.0) incubated with total proteins extracted from the shell of M. coruscus [8] for 4 h at 4°C, and then washed with elution buffer (20 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole, pH 8.0). Eluted protein samples were analyzed using LC-MS /MS after digestion with trypsin. LC-MS/MS experiments were performed on a Q Exactive Plus MS coupled with an Easy nLC (Thermo Scientific). The MS data were analyzed using MaxQuant software (version 1.6.1.0) and searched against the mantle transcriptome database of M. coruscus (accession number: SRX792025) [8] . The results retrieved from the database search were filtered and exported with a < 1% false discovery rate (FDR) at the peptide-spectrum-matched level, and protein level, respectively. The Ni-NTA beads without binding to rVDCP were used as a control, and the identified nonspecific proteins pulled down by blank Ni-NTA beads were removed accordingly from the list of identified protein partners of rVDCP.
After binding to rVDCP, the Ni-NTA beads were washed with binding buffer (20 mM Tris-HCl, 150 mM NaCl, and 10 mM imidazole, pH 8.0) incubated with total proteins extracted from the shell of M. coruscus [8] for 4 h at 4°C, and then washed with elution buffer (20 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole, pH 8.0). Eluted protein samples were analyzed using LC-MS /MS after digestion with trypsin. LC-MS/MS experiments were performed on a Q Exactive Plus MS coupled with an Easy nLC (Thermo Scientific). The MS data were analyzed using MaxQuant software (version 1.6.1.0) and searched against the mantle transcriptome database of M. coruscus (accession number: SRX792025) [8] . The results retrieved from the database search were filtered and exported with a < 1% false discovery rate (FDR) at the peptide-spectrum-matched level, and protein level, respectively. The Ni-NTA beads without binding to rVDCP were used as a control, and the identified nonspecific proteins pulled down by blank Ni-NTA beads were removed accordingly from the list of identified protein partners of rVDCP.
3
Identifying Protein-Protein Interactions via Ni-NTA Affinity Purification and LC-MS/MS
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Ni-NTA beads (Sangon, China) were used to binding the rPDCP-1 protein containing the His6 tag. After binding with rPDCP-1, the Ni-NTA beads were washed with binding buffer (20 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0), incubated with total proteins extracted from the shell of M. coruscus (Liao et al., 2015 (link)) for 4 h at 4°C, and then washed with elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0). Eluted protein samples were analyzed by LC-MS/MS after digestion by trypsin. LC-MS/MS experiments were performed on a Q Exactive Plus MS coupled with an Easy nLC (Thermo Scientific). The MS data were analyzed using MaxQuant software (version 1.6.1.0.) and searched against the mantle transcriptome database of M. coruscus (Accession: SRX792025 ) (Liao et al., 2015 (link)). The database search results were filtered and exported with a < 1% false discovery rate (FDR) at the peptide-spectrum-matched level and protein level, respectively.
4
Identifying TLP-1 Interactors in Mollusk Shell Matrices
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A His‐tag affinity pull‐down assay was performed to identify the protein partners of TLP‐1 in the shell matrices of M. coruscus using the protocol of Jiang et al. [33 ]. Ni‐NTA beads (Sangon, Shanghai, China) were used for binding rTLP‐1 at the His6 tag contained in the sequence, and the beads were washed with binding buffer (20 mm Tris/HCl, 150 mm NaCl and 10 mm imidazole at pH 8.0) and then incubated for 4 h with total proteins extracted from the shell matrices of M. coruscus at 4 °C and then washed with elution buffer (20 mm Tris/HCl, 300 mm NaCl and 300 mm imidazole at pH 8.0). The eluted fractions were analysed by LC‐MS/MS after digestion by trypsin. The LC‐MS/MS experiments were performed on a Q‐Exactive Plus MS spectrometer coupled with an Easy‐nLC chromatograph (Thermo Scientific, Waltham, Massachusetts, USA). The MS data were analysed using maxquant software (version 1.6.1.0., Max‐Planck Institute of Biochemistry, Netherlands, Germany) and searched against the mantle transcriptome database of M. coruscus (Accession: SRX792025) [6 ]. The database search results were filtered and exported based on a < 1% false discovery rate (FDR) at the peptide‐spectrum‐matched level and protein level.
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