Chemoenzymatic labeling and biotinylation of proteins in total cell lysates were carried out as described previously (14 (link), 47 (link)). In brief, proteins (200 μg) were labeled utilizing the Click-iT O-GlcNAc Enzymatic Labeling System (Invitrogen). The permissive mutant β-1,4-galactosyltransferase (GalT) is responsible for the transfer of azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues on target proteins. Modified proteins were detected utilizing the Click-iT Biotin Protein Analysis Detection Kit protocol (Invitrogen). Biotinylated proteins were resolubilized in binding buffer (0.1 M phosphate, 0.15 M NaCl, 0.1% SDS, 1% NP-40, pH 7.2). Appropriate amount of streptavidin resin (Thermo) was added to incubate with the mixture overnight at 4 °C. The streptavidin-bound complex was washed with binding buffer. Following the removal of supernatants, pellets were eluted by boiling with loading buffer (2% SDS, 10% glycerol, 2.5% 2-mercaptoethanol, 62.5 mM Tris–HCl, pH 6.8) and analyzed by WB.
Streptavidin resin
Manufactured by Thermo Fisher Scientific
Streptavidin resin is a solid support material used for affinity purification and capture of biotinylated molecules. It consists of streptavidin, a bacterial protein, covalently bound to a cross-linked agarose matrix. The streptavidin-biotin interaction is one of the strongest non-covalent bonds in nature, making this resin a powerful tool for isolating and concentrating targeted biomolecules.
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Lab products found in correlation
Click it o glcnac enzymatic labeling system, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Hitrap q hp, by GE Healthcare (1 mentions)
Bl21 codonplus de3 ripl, by Agilent Technologies (1 mentions)
Hitrap, by Cytiva (1 mentions)
6 protocols using streptavidin resin
1
Chemoenzymatic Labeling and Biotinylation of Proteins
2
Purification of Recombinant Human Thrombin
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The S195M mutant and WT human thrombin were expressed and refolded from E. coli as previously described8 ,10 (link). After initial refolding and initial purification, the S195M thrombin was mixed with WT thrombin at a ratio of 1:30 WT:S195M and was left to rock for 12–16 h at 30 °C to convert the meizothrombin species formed to α-thrombin. After activation, the WT α-thrombin was removed by addition of biotinyl-PPACK (Haematologic Technologies) then captured on streptavidin resin (Thermo Scientific) and removed. The α-form of the S195M thrombin was isolated from other forms by chromatography on a 10/100 GL MonoS cation exchange column (GE Healthcare Life Sciences) using a gradient of 100 mM–500 mM NaCl in 25 mM phosphate pH 6.5. This method of thrombin purification has been shown to result in > 95% α-thrombin, and previous NMR analysis of isotopically labeled thrombin prepared this way demonstrated that the species present was α-thrombin10 (link),18 (link).
3
Biotinylated FIT2 Peptide Pulldown
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Biotinylated cytosolic FIT2 peptides (Table S1 ) were synthesized by GL Biochem and dissolved in DMSO. Strep-tag cleaved septin hexamer (1 µM) and peptides (20 µM) were used for pull-down assays in a buffer containing 50 mM Tris-Cl, 150 mM KCl, 2 mM MgCl2, and 2 mM GTP. The protein-peptide mixture was incubated with streptavidin resin (Thermo Fisher Scientific; Pierce) for 1 h. The precipitates were washed and analyzed by SDS-PAGE and Coomassie blue staining.
4
Enzymatic Labeling of O-GlcNAcylated PRPS1
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O-GlcNAcylation of PRPS1 using the enzymatic labeling method was carried out as described previously37 (link),54 (link)–56 (link). In brief, 400 μg of total cell or tissue lysate was labeled according to the Invitrogen Click-iT O-GlcNAc enzymatic labeling protocol (Thermo Fisher Scientific, C33368). Enzymatic labeled proteins were conjugated with an alkyne-biotin compound based on Invitrogen Click-iT protein analysis detection kit protocol (Thermo Fisher Scientific, C33372). The biotinylated proteins were precipitated by streptavidin resin (Thermo Fisher Scientific, 20353) and then eluted in a loading buffer containing 20 mM biotin (Sigma-Aldrich, B4501) by boiling. The immunoblot intensities of PRPS1 in elution and in input were measured by ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/ ) to quantify the PRPS1 O-GlcNAcylation level for each sample.
5
RNA Isolation from Nisin-Induced Bacterial Cells
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Bacterial cells (100 ml) were collected after 2–3 hr of nisin induction of RNA expression and disrupted in 800 μl of CB500 solution (20 mM Tris–HCl, pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 1 mM PMSF) by vortexing (30 s, rest for 1 min, 24 cycles) using 500 μl of 0.1 mm ice-cold glass beads (Sigma). Lysates were cleared by centrifugation at 14,000 rpm for 25 min. To pull down RNAs, ~500 μl of cleared lysate was incubated with 100 μl of streptavidin resin (Thermo Scientific) that was equilibrated with CB500. The resin was washed eight times with 1 ml CB500 buffer. RNAs bound to resin were eluted with 400 μl of 5 mM biotin for 1 hr and were extracted from the eluates. RNA identities were determined by using reverse transcription primer extensions.
6
Purification of Recombinant Proteins
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All proteins were expressed in BL21-CodonPlus (DE3)-RIPL (Agilent) or BL21 E. coli strains (NEB). CytATL, cytATL(R232Q), and MBP-cyclin B∆90 were purified, as described previously (Wang et al., 2013 (link)). CytATL-GFP, cytATL(R232Q)-GFP and all cytLnp proteins except cytLnp-SBP were isolated using a Ni-NTA resin (Thermo Scientific), followed by anion-exchange chromatograph (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 200, GE Healthcare). CytLnp-SBP was purified using a Ni-NTA resin, followed by purification on a streptavidin resin (Thermo Scientific) and gel filtration (Superdex 200, GE Healthcare). The proteins were snap-frozen in 20 mM HEPES pH 7.5, 150 mM KCl, 250 mM sucrose, and 1 mM dithiothreitol (DTT). For the gel filtration analysis in Figure 11—figure supplement 1F , 400 µg of purified His6-cytLnp/-N1/-N2/-C in 200 µL were injected onto a Superdex 200 column in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, and 1 mM DTT.
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