Plasmids were transformed into Lemo21 (DE3) cells, and used directly to inoculate 50 mL of auto-induction media (TBII supplemented with 0.5 % w/v glucose, 0.05% w/v glycerol, 0.2% w/v lactose monohydrate, and 2 mM MgSO4, 50 ug/mL kanamycin sulfate). The cultures were incubated at 37 °C for 20–24 h, before harvesting the cells by centrifugation (4,000 x g, 5 min). Cells were resuspended in 10 mL of lysis buffer (100 mM Tris, 150 mM NaCl, pH 8, 0.1 mg/mL lysozyme, 0.01 mg/mL DNAse I, 1 mM PMSF) and lysed by sonication. The insoluble fraction was cleared by centrifugation (16,000 x g for 45 min), and the proteins were purified from the soluble fraction by affinity chromatography using Strep-Tactin XT Superflow High-Capacity resin (IBA Lifesciences). Elutions were performed with 100 mM Tris, 150 mM NaCl, 50 mM biotin, pH 8, and the proteins were further purified by size-exclusion chromatography using a Superdex 200 10/300 increase column equilibrated with 20 mM sodium phosphate, 100 mM NaCl, pH 7.4, 0.05% v/v Tween 20.
Strep tactin xt superflow high capacity resin
Manufactured by IBA Lifesciences
Sourced in Germany
Strep-Tactin XT Superflow high-capacity resin is a versatile affinity chromatography resin used for the purification of Strep-tagged proteins. It is designed for high-throughput protein purification with a binding capacity of up to 100 mg of Strep-tagged protein per milliliter of resin.
Automatically generated - may contain errors
Lab products found in correlation
Elution buffer, by Merck Group (2 mentions)
Centrifugal filter, by Merck Group (2 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (2 mentions)
Nickel sepharose excel resin, by GE Healthcare (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Desalting column, by GE Healthcare (1 mentions)
Superdex 75 gel filtration column, by GE Healthcare (1 mentions)
5 protocols using strep tactin xt superflow high capacity resin
1
Recombinant Protein Purification from Lemo21 (DE3) Cells
2
Expression and Purification of Trimeric SARS-CoV-2 Spike
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression and purification of the S trimers were carried out as previously described14 (link). Briefly, Expi293F cells (Gibco) were transiently transfected with the plasmids encoding different trimeric S proteins using Expifectamine 293 Transfection kit (Life Technologies, USA) according to manufacturer’s instructions. Cell culture supernatants were collected 3 d later and purified using Strep-Tactin XT Superflow high-capacity resin (IBA Lifesciences). After washing with 10-bed volumes of wash buffer (20 mM Tris, 200 mM NaCl, pH 8.0), the S trimers were eluted by the elution buffer (wash buffer with 50 mM biotin) (Sigma). After elution, the elution buffer was replaced with sodium citrate buffer (0.1 M sodium citrate, pH 5.5) using a centrifugal filter (Millipore), immediately followed by negative staining analysis. For expression and purification of soluble ACE2s or RBD proteins, the procedures were similar to above except that the elution buffer was replaced with the wash buffer (20 mM Tris, 200 mM NaCl, pH 8.0).
3
Expression and Purification of Trimeric SARS-CoV-2 Spike
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression and purification of the S trimers were carried out as previously described14 (link). Briefly, Expi293F cells (Gibco) were transiently transfected with the plasmids encoding different trimeric S proteins using Expifectamine 293 Transfection kit (Life Technologies, USA) according to manufacturer’s instructions. Cell culture supernatants were collected 3 d later and purified using Strep-Tactin XT Superflow high-capacity resin (IBA Lifesciences). After washing with 10-bed volumes of wash buffer (20 mM Tris, 200 mM NaCl, pH 8.0), the S trimers were eluted by the elution buffer (wash buffer with 50 mM biotin) (Sigma). After elution, the elution buffer was replaced with sodium citrate buffer (0.1 M sodium citrate, pH 5.5) using a centrifugal filter (Millipore), immediately followed by negative staining analysis. For expression and purification of soluble ACE2s or RBD proteins, the procedures were similar to above except that the elution buffer was replaced with the wash buffer (20 mM Tris, 200 mM NaCl, pH 8.0).
4
Purification of SARS-CoV-2 Receptor-Binding Domain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All constructs were purified essentially as previously described31 (link). A first immobilized affinity chromatography (IMAC) step was performed using Nickel Sepharose Excel resin (GE Healthcare). Columns were equilibrated with equilibration buffer (50 mM NaPO4 pH 7.8, 300 mM NaCl) and supernatants were loaded at 3 mL/min. Columns were washed once with 50 mM NaPO4 pH 7.8, containing 300 mM NaCl and 10 mM imidazole and eluted with the same buffer containing 300 mM imidazole. Wild type RBD and its variants were further purified using Superdex75 gel filtration column (GE Healthcare) except for the Omicron variant that was further purified by anti-FLAG chromatography55 (link) prior to Superdex75 gel filtration. This is necessary to separate monomers from dimers. The IMAC-purified ACE2 protein was loaded on Strep-Tactin XT Superflow high-capacity resin (IBA Lifesciences, Germany) according to the manufacturer’s instructions. Buffer exchange was done with desalting columns (GE Healthcare) with PBS. Purified proteins were quantified using a NanoDrop Spectrophotometer (ThermoFisher). Finally, SDS-PAGE and total protein staining (Coomassie Blue) were performed using standard methods.
5
Recombinant Protein Purification from Lemo21 (DE3) Cells
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!