Cells were resuspended in buffer C (25 mM Tris–HCl pH 7.5, 200 mM NaCl, 10% glycerol, and 0.5 mM TCEP) supplemented with 5 mM imidazole, 0.05% Tween-20, and cOmplete Protease-Inhibitor-Cocktail (Roche). Cells were lysed by sonication, cleared by centrifugation (20 min, 20,000 × g, 4°C), and the clarified lysate was purified on Ni-NTA resin (QIAGEN) eluting in buffer C supplemented with 200 mM imidazole. The eluted protein was incubated with Strep-Tactin Sepharose (IBA Lifesciences) beads, washed with 10 column volumes of buffer B, and eluted with buffer C supplemented with 5 mM Desthiobiotin. Strep-Tactin purified mPSF complexes were concentrated in centrifugal filter (Amicon Ultra-15, MWCO 300 kDa, Merck Millipore) to approximately 0.5 mg ml−1. To account for impurities, mPSF complex concentrations were assessed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and adjusted accordingly (Figure S1C), aliquoted, flash frozen, and stored at −80°C. For use in pull-down analysis, CPSF complexes were used directly after Strep-Tactin purification.
Strep tactin sepharose
Manufactured by IBA Lifesciences
Sourced in Germany
Strep-Tactin Sepharose is a resin-based affinity chromatography medium used for the purification of Strep-tag fusion proteins. It consists of Sepharose beads coupled with the Strep-Tactin protein, which has a high affinity for the Strep-tag. This resin can be used to capture and purify Strep-tagged proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta agarose, by Qiagen (3 mentions)
Amicon ultra 15, by Merck Group (2 mentions)
Ni nta resin, by Qiagen (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Surebeads protein g magnetic beads, by Bio-Rad (2 mentions)
Lmg194 bacterial cells, by Thermo Fisher Scientific (2 mentions)
Emulsiflex c3 homogenizer, by Avestin (1 mentions)
Sephadex g 25 resin, by GE Healthcare (1 mentions)
Sephadex g 25, by Merck Group (1 mentions)
Atto 647n maleimide, by ATTO-TEC (1 mentions)
Amicon ultra centrifugal filters 10k, by Merck Group (1 mentions)
Lobind tube, by Eppendorf (1 mentions)
Superdex 200, by Cytiva (1 mentions)
Protease inhibitor mix, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Ni nta, by Qiagen (1 mentions)
Desthiobiotin, by IBA Lifesciences (1 mentions)
T7 express, by New England Biolabs (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Glutathione sepharose, by Cytiva (1 mentions)
29 protocols using strep tactin sepharose
1
Purification of mPSF Protein Complex
2
Overexpression and Purification of Strep-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were overproduced in E. coli BL21. Overexpression strains were cultivated in 1 l LB broth (containing 100 μg/ml ampicillin) at 37°C and 250 rpm. Protein expression was induced by addition of 1 mM IPTG at OD600 = 0.5. Cultivation was continued over night at 16°C and 200 rpm. Cells were harvested by centrifugation (11.325 x g, 10 min, 4°C) and the cell pellet was washed once in 20 ml ZAP buffer. Afterwards, the cell pellet was resuspended in 20 ml ZAP buffer containing 1 mM PMSF. Cells were disrupted using an EmulsiFlex C3 homogenizer (Avestin Europe GmbH), cell debris was removed by centrifugation (4.581 x g, 10 min, 4°C) and the lysate was cleared in an additional passage through a filter (0.45 μm pore size). Strep-tagged proteins were purified using affinity chromatography and Strep-Tactin Sepharose (IBA Lifesciences, Germany) according to the manufacturer’s instructions. Fractions containing purified proteins were pooled, aliquoted and stored at -20°C.
3
Purification of DHFR Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All
DHFR variants were constructed, overexpressed, and purified using
standard molecular biology techniques,50 (link),55 (link) as described
in full detail in theSupporting Information section 5.2. Briefly, the DHFR4S DNA construct was built
from the pT7-SC1 plasmid containing the DHFR-tag construct50 (link) by inserting an additional alanine residue at
position 175 (located in the fusion tag) with site-directed mutagenesis.
All other variants were derived—again using site-directed mutagenesis—either
directly from DHFR4S or from a variant thereof. The plasmids
of each DHFR variant were used to transform E. cloni EXPRESS BL21(DE3)
cells (Lucigen, Middleton, USA), and the DHFR proteins they encode
were overexpressed overnight at 25 °C in a liquid culture. After
the bacterial cells were harvested by centrifugation, the overexpressed
proteins were released into solution through lysis—using a
combination of at least a single freeze–thaw cycle, incubation
with lysozyme, and probe–tip sonification. Finally, the DHFR
proteins were purified from the lysate with affinity chromotography
with Strep-Tactin Sepharose (IBA Lifesciences, Goettingen, Germany),
aliquoted, and stored at −20 °C until further use.
DHFR variants were constructed, overexpressed, and purified using
standard molecular biology techniques,50 (link),55 (link) as described
in full detail in the
from the pT7-SC1 plasmid containing the DHFR-tag construct50 (link) by inserting an additional alanine residue at
position 175 (located in the fusion tag) with site-directed mutagenesis.
All other variants were derived—again using site-directed mutagenesis—either
directly from DHFR4S or from a variant thereof. The plasmids
of each DHFR variant were used to transform E. cloni EXPRESS BL21(DE3)
cells (Lucigen, Middleton, USA), and the DHFR proteins they encode
were overexpressed overnight at 25 °C in a liquid culture. After
the bacterial cells were harvested by centrifugation, the overexpressed
proteins were released into solution through lysis—using a
combination of at least a single freeze–thaw cycle, incubation
with lysozyme, and probe–tip sonification. Finally, the DHFR
proteins were purified from the lysate with affinity chromotography
with Strep-Tactin Sepharose (IBA Lifesciences, Goettingen, Germany),
aliquoted, and stored at −20 °C until further use.
4
Site-specific labeling of Get3 and Mini-Get1/2 proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Get3 with a ybbR tag (DSLEFIASKLA) inserted between residues S110 and D111 was labeled with BDP-maleimide (Thermo Fisher Scientific) or with a 1:1 mixture of ATTO550-maleimide (GE Healthcare) and ATTO 647N-maleimide (ATTO-TEC) via Sfp-catalyzed incorporation of dye-CoA conjugates (Rao et al., 2016 (link); Chio et al., 2019 (link), 2017b (link)). The C-terminal His6-tagged Sfp was removed through Ni-NTA affinity chromatography. The excess dye–CoA conjugates were further removed through gel filtration using a Sephadex G-25 (Sigma-Aldrich) column. Get3ATTO550/ATTO647N-TA complex and Get3BDP-TACm were generated by in vitro translation of TA substrate 3xStrep-SUMOnc-Bos1-opsin in E. coli S30 cell extract (Rao et al., 2016 (link)) supplemented with Get3 variants and untagged T7 RNA polymerase and/or untagged CmRS (for generating Get3BDP-TACm). The resulting Get3-TA complex was purified using Strep-Tactin Sepharose (IBA Life Sciences; Rao et al., 2016 (link); Chio et al., 2019 (link)).
Mini-Get1/2 was labeled with TMR-5-maleimide (Invitrogen) via thiol-maleimide reaction on an engineered single cysteine at residue S77 of Get1 or residue T34 of Get2. The labeling reaction was quenched with 1 mM DTT, and excess dye was removed using a PD-10 column packed with Sephadex G-25 resin (GE Healthcare).
Mini-Get1/2 was labeled with TMR-5-maleimide (Invitrogen) via thiol-maleimide reaction on an engineered single cysteine at residue S77 of Get1 or residue T34 of Get2. The labeling reaction was quenched with 1 mM DTT, and excess dye was removed using a PD-10 column packed with Sephadex G-25 resin (GE Healthcare).
5
WNV Envelope Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture supernatants of transfected 293 T cells were harvested and treated with 1% Triton X-100. The supernatants were immunoprecipitated or pulled down using an anti-WNV E antibody (Millipore) or Strep-Tactin Sepharose (IBA Lifesciences, Gottingen, Germany). Immunoprecipitation was performed by incubating the supernatants at 4 ℃ for 3 h with 20 μL of antibody-coupled Surebeads Protein G Magnetic Beads (Bio-Rad, Hercules, CA). The supernatants were next incubated with Strep-Tactin Sepharose at 4 ℃ for 2 h. The reaction and elution steps were performed following the manufacturer’s instructions. The precipitated protein was separated using SDS-PAGE and analyzed by immunoblotting as described above.
6
Hsf1-Ssa1 Complex Purification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified Hsf1-StrepTag II-Ssa1 complexes were incubated with different concentrations of ATP in LWB150 and was bound to Strep-Tactin Sepharose (IBA Life Sciences). After extensive washing with LWB150, bound protein was eluted using LWB150 + 2.5 mM desthiobiotin. Samples were separated by SDS-PAGE and visualized by Pierce(TM) Silver Staining Kit (Thermo Fisher).
7
Luciferase Protein Purification
8
Purification of Phytochrome Photoreceptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BphP1 and BphP1-mRuby2 proteins with polyhistidine tags on the N-terminus were expressed in LMG194 bacterial cells (Life Technologies-Invitrogen) containing a pWA23h plasmid encoding heme oxygenase for biliverdin synthesis in Escherichia coli18 (link). The bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 6-8 h followed by an induction of the protein expression by adding of 0.002% arabinose. The proteins were purified using a Ni-NTA agarose (Qiagen). The PpsR2 and PpsR2-mVenus proteins with a Strep-tag-II at the N-terminus and a polyhistidine tag at the C-terminus was expressed in BL21(DE3) bacterial cells grown in LB medium supplemented with ampicillin for 6 h, followed by an induction of a protein expression with 250 μM IPTG. The proteins were first purified with a Ni-NTA agarose (Qiagen) followed by purification with a Strep-Tactin sepharose (IBA Lifesciences).
9
Affinity Purification of SARS-CoV-2 Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 human alveolar epithelial cells were transfected with empty vector in triplicate or with vectors expressing SARS CoV-2 Nsp3ΔTM or PLpro fused to a 2xStrep tag at the N-terminus (4 replicates each). 12hr later, cells were stimulated with 50 ng/ml IFN-a (Cell Signalling Technologies) for 36hr. Cells were lysed in ice-cold IP buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.5% NP-40, 1 mM AEBSF) and clarified by centrifugation. Viral proteins were affinity purified from 4 mg lysates by tumbling with 40 μl Strep-Tactin Sepharose (IBA Life Sciences) for 90 min at 4°C. Beads were washed two times with IP buffer containing 500 mM NaCl and 0.5% NP-40 and twice more with IP buffer without detergent. 90% of the final wash was transferred to a fresh LoBind tube (Eppendorf) for processing for LC-MS/MS analysis. The remaining 10% was reserved to confirm viral protein expression and pulldown by Western blot for Nsp3. Efficient IFN-a stimulation was confirmed by blotting lysates for ISG15.
10
Ubiquitin Immunoprecipitation of WNV-Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transfection of pCXSN-flag-ubiquitin, SH-SY5Y cells infected with WNV were washed with PBS and then harvested in 1% Triton buffer. The lysates were immunoprecipitated with 1 μg anti-Flag antibody. Immunoprecipitation was performed by incubating the cell lysates at 4°C for 3 h with 10 μl antibody-coupled Surebeads Protein G Magnetic Beads (Bio-Rad) according to the manufacturer’s protocol. The lysates from SH-SY5Y cells transfected with each plasmid were incubated with Strep-Tactin Sepharose (IBA Lifesciences, Gottingen, Germany) at 4°C for 3 h. The reaction and elution were performed according to the manufacturer’s instructions. The precipitated protein complexes were separated by SDS-PAGE and analysed by immunoblotting.
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!