For protein expression and purification the EnPresso™ Tablet Cultivation Set (BioSilta, Oulu, Finnland) and HaloTag® Protein Purification System (Promega) were used, respectively. To induce protein expression, a transformed culture of KRX competent cells (Promega) at an optical density of 9-13 at 600 nm was supplemented with a “booster solution” (EnZ I’m and 0.05% rhamnose). After centrifugation for 10 min at 5600 rpm and 4°C, the cell pellet was resuspended in HaloTag® Protein Purification buffer (50 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.005% IGEPAL CA-630; Promega), 10 mg/ml lysozyme (Sigma-Aldrich) and RQ1 RNase free DNase (Promega) and disrupted by sonication at 60% power for 45 s on ice (Sonicator UP50H; Dr. Hielscher GmbH, Teltow, Germany). The proteins were purified from the sonicated cell lysates according to the manufacturer’s recommendations (Promega). Briefly, lysates were incubated with HaloLink™ resin, followed by washing with HaloTag® Protein Purification buffer and cleavage with TEV Protease Cleavage Solution (HaloTag® Protein Purification buffer supplemented with 1/16 volume TEV protease) on a rotator (NeoLab, Heidelberg, Germany) for 1 h at RT. After centrifugation 50 μl of 50% HisLink™ resin was added and incubated on the rotator for 20 min at RT. The supernatant contained the recombinant proteins.
Halotag protein purification system
Manufactured by Promega
Sourced in Germany
The HALOTAG® Protein Purification System is a laboratory equipment used for the purification of recombinant proteins expressed in various host cells. The system utilizes a HaloTag® protein tag that binds to a solid support matrix, allowing for the capture and purification of the tagged protein.
Automatically generated - may contain errors
Lab products found in correlation
Hek293 and e coli krx, by Promega (3 mentions)
Krx competent cells, by Promega (1 mentions)
Igepal ca 630, by Promega (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Halolink resin, by Promega (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Gsk 3β, by New England Biolabs (1 mentions)
9 protocols using halotag protein purification system
1
Recombinant Protein Expression and Purification
2
Cloning and Purification of Human eIF5B
3
Mass Spectrometric Analysis of OgLuc Variants
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Example 35
To determine if the OgLuc variants undergo any post translation modifications when expressed in mammalian cells, the 9B8 and L27V variants were expressed in both mammalian cells and E. coli and analyzed via mass spectrometry (MS).
9B8 and L27V variants were expressed as N-terminal HALOTAG® fusions (pFN18K for E. coli; pFN21K for HEK293 cells) in HEK293 and E. coli KRX (Promega Corp.) cells and purified using the HALOTAG® Protein Purification System (Promega Corp.) according to the manufacture's instructions. Approximately 5 pmols of purified enzyme was analyzed via LC/MS using a C4 column (Waters Xbridge BEH300, 3.5 μm) interfaced to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data was acquired from 600-2000 m/z using the LTQ for detection and processed using the MagTran v1.03 software (Zhang et al., J. Am. Soc. Mass Spectrom., 9:225-233 (1998)). Both purified enzymes had an experimentally determined mass of 19,666 Da, compared to a calculated mass of an un-modified OgLuc variant, i.e., absent of any post translational modifications, of 19,665 Da.
4
Mass Spectrometry Analysis of OgLuc Variants
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Example 35
To determine if the OgLuc variants undergo any post translation modifications when expressed in mammalian cells, the 9B8 and L27V variants were expressed in both mammalian cells and E. coli and analyzed via mass spectrometry (MS).
9B8 and L27V variants were expressed as N-terminal HALOTAG® fusions (pFN18K for E. coli; pFN21K for HEK293 cells) in HEK293 and E. coli KRX (Promega Corp.) cells and purified using the HALOTAG® Protein Purification System (Promega Corp.) according to the manufacture's instructions. Approximately 5 pmols of purified enzyme was analyzed via LC/MS using a C4 column (Waters Xbridge BEH300, 3.5 μm) interfaced to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data was acquired from 600-2000 m/z using the LTQ for detection and processed using the MagTran v1.03 software (Zhang et al., J. Am. Soc. Mass Spectrom., 9:225-233 (1998)). Both purified enzymes had an experimentally determined mass of 19,666 Da, compared to a calculated mass of an un-modified OgLuc variant, i.e., absent of any post translational modifications, of 19,665 Da.
5
FOXA2 Protein Purification from 293Ts
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293Ts were transfected with pFN21A-FOXA2. Purification was completed following Promega’s Halotag Protein Purification System. Briefly, 48 hours following transfection, cells were harvested, lysed and gently sonicated four times on Branson Sonifier at 10% amplitude for 15 seconds. Sample was diluted 1:3 with protein purification buffer (1X PBS, 1mM DTT, 0.0005% NP-40) and centrifuged to remove debris. Halo-Resin was washed in purification buffer, added to lysate and incubated at 4C overnight. After incubation resin was washed and FOXA2 protein was cleaved via the addition of TEV-protease during an over night incubation at 4C. Purified protein was assessed via commassie blue gel and western blot.
6
ETS1 Phosphorylation by GSK3β In Vitro
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Halo-tag ETS1 was purified with Halo Tag Protein purification system (Promega). In briefly, Halo-ETS1 transfected 293 cell pellets were lysed in Halo Tag purification buffer (1x PBS, 1mM DTT, 0.005% IGEPAL-CA630 (Sigma) and protease inhibitor). After freeze-thawing three times, the supernatants were added into HaloLink resin (Promega) according manufacturer instructions. Halo-ETS1 was released from the resin by using HaloTEV enzyme to cleavage TEV cutting site between Halo tag and ETS1 protein. The purified ETS1 protein was incubated with GSK3β (New England BioLabs) in GSK3 reaction buffer (20mM Tris-HCl, 10mM MgCl2, 2mM DTT and 200μM ATP) at 3° C for 30 mins. Finally, The phosphorylation levels of purified ETS1 were performed by western blot with anti-phosphoserine/threonine antibody (Cell signaling). The reagents and antibodies used for in vitro kinase assay are shown in Supplementary Table 2 .
7
Mass Spectrometry Analysis of OgLuc Variants
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Example 35
To determine if the OgLuc variants undergo any post translation modifications when expressed in mammalian cells, the 9B8 and L27V variants were expressed in both mammalian cells and E. coli and analyzed via mass spectrometry (MS).
9B8 and L27V variants were expressed as N-terminal HALOTAG® fusions (pFN18K for E. coli; pFN21K for HEK293 cells) in HEK293 and E. coli KRX (Promega Corp.) cells and purified using the HALOTAG® Protein Purification System (Promega Corp.) according to the manufacture's instructions. Approximately 5 pmols of purified enzyme was analyzed via LC/MS using a C4 column (Waters Xbridge BEH300, 3.5 μm) interfaced to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data was acquired from 600-2000 m/z using the LTQ for detection and processed using the MagTran v1.03 software (Zhang et al., J. Am. Soc. Mass Spectrom., 9:225-233 (1998)). Both purified enzymes had an experimentally determined mass of 19,666 Da, compared to a calculated mass of an un-modified OgLuc variant, i.e., absent of any post translational modifications, of 19,665 Da.
8
FOXA2 Protein Purification from 293Ts
9
Purification of NOTCH3 EGF-like Domains
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We used transient transfection of human embryonic kidney 293T cells and the HaloTag Protein Purification System (Promega GmbH, Mannheim, Germany) for the purification of recombinant NOTCH3 fragments consisting of the first 5 epithelial growth factor-like repeat domains (online-only Data Supplement).
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