Ni chelating sepharose affinity chromatography

Manufactured by GE Healthcare

Ni-chelating Sepharose affinity chromatography is a laboratory equipment used for the purification and separation of proteins. It utilizes the specific binding interaction between nickel ions (Ni2+) immobilized on a Sepharose resin and proteins containing histidine-tags or other nickel-binding motifs. This technique allows for the selective capture and isolation of the target proteins from complex mixtures.

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Lab products found in correlation

Q ion exchange chromatography, by GE Healthcare (2 mentions) Pet32a, by Thermo Fisher Scientific (1 mentions) Ny eso 1, by Thermo Fisher Scientific (1 mentions) Cpg odn, by Thermo Fisher Scientific (1 mentions) Endotoxin free ovalbumin ova, by InvivoGen (1 mentions)

Close spelling variants of this product

Ni Sepharose affinity chromatography Ni-chelating chromatography Ni-chelating Sepharose Ni affinity chromatography Ni-Sepharose chromatography Chelating Sepharose Ni Sepharose

2 protocols using ni chelating sepharose affinity chromatography

Production and Purification of NY-ESO-1 Protein

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The production and purification for NY-ESO-1 protein were presented by the following steps. First, we assembled a bacterial expression plasmid PET32a (Invitrogen) and NY-ESO-1 (Invitrogen). Then we injected PET32a-NY-ESO-1 into the E. coli strain BL21 (DE3)-bearing to make it recombine, and induced it with IPTG for protein production. Finally we lysed the E. coli using a high-pressure homogenizer (APV 2000, Lubeck, Germany) to attain crude NY-ESO-1 protein (Trx-NY-ESO-1). Here are four steps for the purification of NY-ESO-1 protein, which included Ni-chelating Sepharose affinity chromatography (GE Healthcare, Piscataway, NJ), excision of the Trx-His6-tag, removal of the Trx-His6-tag with second Ni-chelating Sepharose affinity chromatography, and Q-ion-exchange chromatography (GE Healthcare, Piscataway, NJ). The endotoxin level was below standard level.
The preparation of combine vaccine needs the following steps. In short, 20 μg CpG ODN was mixed with 40 μg HH2, and then combined with 125 μg alum, next mixed with 5 μg recombinant NY-ESO-1 protein in PBS with a total volume of 100 μL. The endotoxin levels were approximately 0.02 EU/μg.

Wang H., Huang W., Gao H, & Liu T.T. (2020). NY-ESO-1 Protein Vaccine Combining Alum, CpG ODN, and HH2 Complex Adjuvant Induces Protective and Therapeutic Anti-Tumor Responses in Murine Multiple Myeloma. OncoTargets and therapy, 13, 8069-8077.

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Synthesis and Purification of DP2-11 Peptide

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DP2-11 was synthesized by using fluorenyl-methyloxycarbonyl (Fmoc) chemistry at the Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China). The synthesized peptides were purified by high-performance liquid chromatography to obtain 95% purity, and their molecular weights were confirmed by mass spectrometry. The peptide powder was reconstituted in sterile water at a concentration of 10 mg/ml and stored at −20 °C. CpG ODN (murine TLR9 agonist, 5′-tccatgacgttcctgacgtt-3′) contains a full phosphorothioate backbone and was synthesized by Invitrogen Life Technologies. The CpG ODN powder was dissolved in sterile water at a concentration of 5 mg/ml and then stored at −20 °C until use. The aluminum hydroxide gel adjuvant (alum) was obtained from Brenntag Biosector, Frederikssund, Denmark. The NY-ESO-1 antigen, in which the endotoxin level was approximately 0.02 EU/μg, was purified via four process steps including Ni-chelating Sepharose affinity chromatography (GE Healthcare, Piscataway, NJ), excision of the Trx-His6-tag, removal of the Trx-His6-tag with second Ni-chelating Sepharose affinity chromatography, and Q-ion-exchange chromatography (GE Healthcare, Piscataway, NJ). Endotoxin-free ovalbumin (OVA) was purchased from InvivoGen.

Tian Y., Hu Q., Zhang R., Zhou B., Xie D., Wang Y., Zhang X, & Yang L. (2021). Rational design of innate defense regulator peptides as tumor vaccine adjuvants. NPJ Vaccines, 6, 75.

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