1.65 × 106 FL3 cells were seeded in a P10 dish and transfected 24 h later with 12 μg total DNA (2 μg Twin-Strep-Tag-RBP expression vector and 10 μg pcDNA3 PL). Forty-eight hours later, cells were washed with PBS and placed on ice. One milliliter cold lysis buffer (50 mM TRIS-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100, 2% Hexane-1,6-diol, 1 pill Complete pr. 10 mL) was added per plate, and cells were scraped off and transferred to an Eppendorf tube. Samples were mixed and spun (13,000 RPM, 15 min at 4 °C), and 50 μL supernatant was transferred to 50 μL SDS load buffer while 100 μL supernatant was transferred to 0.5 mL Trizol (INPUT). Eight hundred microliters supernatant was incubated with pre-equilibrated MagStrep type 3 XT beads (IBA Life sciences) and rotated for ≥ 2 h at 4 °C. Five hundred microliters supernatant was collected (FT) and samples were washed 4× with 1.5 mL WASH1 buffer (10 mM TRIS-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100) and 2× with WASH2 buffer (10 mM TRIS-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2). During the last wash, sample beads were divided 1:10 (RNA:protein) and added 40 μL SDS load buffer or 0.5 mL Trizol (IP). INPUT, FT, and IP samples were analyzed with western blotting and RT-qPCR.
Magstrep type3 xt beads
Manufactured by IBA Lifesciences
Sourced in Germany
MagStrep "type3" XT beads are paramagnetic beads used for the separation and purification of biomolecules. The beads are coated with Streptavidin, which has a high affinity for biotin-labeled targets. The beads can be easily separated from the reaction mixture using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Bugbuster, by Merck Group (2 mentions)
Strep tactin beads, by IBA Lifesciences (2 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Benzonase, by Merck Group (1 mentions)
7k mwco, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Hek293 flp in t rex cells, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Gelcode blue, by Thermo Fisher Scientific (1 mentions)
Strep tag 2, by Qiagen (1 mentions)
Dylight 800 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Strep tactin hrp conjugate, by IBA Lifesciences (1 mentions)
19 protocols using magstrep type3 xt beads
1
Affinity Purification of RBP Complexes
2
Affinity Purification of Strep-Tagged Proteins
3
Immunoprecipitation of PrPΔGPI and p97 Isoforms
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In 6-cm dishes, HEK293T cells were seeded and co-transfected with PrPΔGPI and p97Wt-mycStrep or p97-EQ-mycStrep. 24 h post-transfection, cells were harvested (1,000g, 5 min), lysed with 500 μl lysis buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris–HCl pH 7.4, 1% Triton X-100, 5% glycerol, 2 mM β-mercaptoethanol supplemented with Complete EDTA-free protease inhibitors and PhosSTOP, Roche), gently resuspended by pipetting, and incubated on ice for 20 min. The lysates were centrifuged at 18,000g for 15 min at 4°C, and the supernatant (input) was collected in separate tubes. MagStrep “type3” XT beads (IBA Lifesciences GmbH) pre-washed with lysis buffer were added to the supernatants and incubated for 2 h at 4°C with gentle rotation. The flowthrough was removed, and the beads were washed thrice with 500 μl lysis buffer. Finally, the beads were boiled with 50 μl 2x Laemmli sample buffer for 5 min to elute the immunocomplex. All samples were fractionated by SDS–PAGE gels and further analyzed through immunoblotting against VCP and PrP.
4
Immunoprecipitation Protocol for Protein Analysis
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For immunoprecipitation, the cells were lysed in PRO-PREP protein extraction solution (iNtRON). Then cell extracts were incubated with MagStrep “type3” XT beads (Cat# 2-4090-002; IBA Lifesciences GmbH, Germany) overnight at 4 °C with constant rotation. Immunocomplexes were washed four times with IP lysis buffer (25 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 5% glycerol), and subsequently resolved by SDS-PAGE followed by western blot analysis.
5
Purification of SARS-CoV-2 NSP3 and NSP4
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HEK 293T cells transiently expressing 2xStrep-NSP3 or NSP4-2xStrep were lysed in a buffer containing 150 mM NaCl, 50 mM HEPES, and 1% deoxy Big CHAP with 1 mM PMSF. Samples were pelleted at high speed to remove unbroken cells and debris, and the supernatant was subject to strep immunopurification using MagStrep “type3” XT beads (Cat#126572-4090-002; IBA Lifesciences) overnight at 4°C. The samples were then washed five times with lysis buffer, and 2xStrep-NSP3 and NSP4-2xStrep were eluted using Buffer E (0.1 M Tris-Cl, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin).
6
Histone Octamers and CAF-1 Binding Assay
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MagStrep Type 3 XT beads (IBA Lifesciences) were preblocked by incubating in binding buffer (50 mM Tris pH 7.6, 350 mM KCl, 2 mM EDTA, 10% glycerol) + 1% BSA at 4 °C overnight. Twenty picomoles each of histone octamers and CAF-1 were premixed in 500 μl of the binding buffer for 1 h at 4 °C, after which 2 μl of a 5% preblocked bead slurry was added, and mixtures incubated at 4 °C with gentle rotation overnight. Beads were immobilized on a magnetic rack and washed 3× with binding buffer containing either 350 mM or 1 M KCl. A final wash was done in BC100 to remove excess salts, and reactions analyzed by Western blot. Band intensities were determined using Image J.
7
Investigating EPRS1 Interactors in TGF-β Signaling
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HEK293T cells were cotransfected with Strep-EPRS1 and each of Flag-TβRI, TβRII, GFP-SMAD2, SMAD3, SMAD4, or SMAD7 were lysed with lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, and protease inhibitor). Cell lysates were incubated with MagStrep type3 XT beads (IBA life sciences, 2-4090-002) for an hour at 4 °C with rotation. Then, the beads were washed three times with the wash buffer 1 (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40) by short vortex. Coprecipitated proteins were dissolved in the SDS sample buffer, denatured for 5 min at 100 °C and separated in the magnetic separator. The supernatants were then subjected to SDS-PAGE.
8
Affinity Purification of Strep-Tagged Proteins
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Cell pellets collected at the end of in vivo translation experiments were lysed by resuspension in 100 μL of BugBuster® (EMD Millipore) and incubation with shaking for 15 min at room temperature. Cell lysates were then diluted in Buffer W (50 mM HEPES pH 8, 150 mM NaCl, 1 mM EDTA) to a final volume equal to 500 μL less the volume of beads, vide infra, and mixed with magnetic Strep-Tactin beads (5% (v/v) suspension of MagStrep “type3” XT beads, IBA Lifesciences), equilibrated in Buffer W. 100 μL of beads were used in the purification of all samples for which a yield is reported (Extended data Table 3 ); all other samples were purified with 20 μL of beads. Samples were then incubated for 30 min at 4 °C with constant gentle inversion. Beads were then pulled down with a magnetic rack, washed with Buffer W (2×500 μL), and eluted with 25 μL of buffer BXT (100 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 mM D-biotin; for PrK incorporation experiments, buffer BXT was made with 50 mM HEPES pH 8 instead of Tris, due to the incompatibility of Tris with copper-catalyzed click conjugation) for 10 min at room temperature with occasional vortexing. Purified proteins were quantified using a Qubit™ Protein Assay Kit (Thermo Fisher).
9
Interaction Analysis of MglA-RomY Complex
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In all experiments involving MglA-His6, MglA-His6 was preloaded with GTP or GDP (44.4μM protein, 22.2mM GTP/GDP) for 30min at RT in buffer C. Subsequently, equimolar amounts of Strep-RomY and MglA-His6, His6-MglB or His6-MalE were incubated for 30min RT in buffer C. Final concentrations: MglA-His6, His6-MglB, His6-MalE, Strep-RomY: 20μM, GTP/GDP 10mM. Where indicated, DSP was added to a final concentration of 200μM for 5min at RT. Next, all reactions were quenched with Tris pH 7.6 added to a final concentration of 100mM and incubated for 15min at RT. Subsequently, 20μl of Strep-Tactin coated magnetic beads (MagStrep ‘type3’ XT beads (IBA-Lifesciences)) previously equilibrated with buffer C were added and samples incubated for 30min RT. The beads were washed 10 times with 1mL buffer C. For experiments with GTP or GDP, buffer C was supplemented with 5mM GTP/GDP. Proteins were eluted with 100μL elution buffer (100 mM Tris pH 8.0, 150mM NaCl, 1mM EDTA, 50mM biotin). Samples were prepared in SDS-PAGE loading buffer (60mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue, 5 mM EDTA) with or without 100mM DTT (final concentration) as indicated. In all SDS–PAGE experiments, equivalent volumes of loading and wash fractions and two-fold more of the elution fraction were loaded and gels stained with Coomassie Brilliant Blue and subsequently analyzed by immunoblotting.
10
Purification of L2N-CVIM Lipopeptides
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To produce L2N-CVIM lipopeptides, 293T cells were transfected with pZeo5-tsa-furin-L2N-CVIM-based plasmids, which express L2N peptides with an N-terminal Twin-Strep-tag and a C-terminal CVIM motif. Specifically, 1 × 106 transfected cells were lysed using 200 μl lysis buffer (PBS with 1% Triton X-100, 1× protease inhibitor cocktail, and 70 mU/ml of BioLock biotin blocking solution) at 48 h posttransfection. The L2N peptides carrying the Twin-Strep-tag were captured by using 1 μl settled MagStrep “type 3” XT beads (IBA Lifesciences) at 4°C for 1 h. After three washes with wash buffer (100 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 1 mM EDTA), the peptides were released from the beads by furin cleavage by incubation with 10 to 20 μl of the 50× concentrated furin-containing supernatant at 37°C for 30 min. The concentration of the eluted peptide can be determined by immunoblotting using JWW-1 antibody with a synthetic L2N peptide of a known concentration. Generally, approximately 2 μg of L2N lipopeptide can be purified from 1 × 106 transfected cells.
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