The diGly-containing peptide enrichment was performed following a procedure published previously172 (link). The diGly monoclonal antibody (Cell Signaling Technology, Cat#5562) (32 μg/IP) was coupled to Protein A Plus Ultralink resin (40 μL slurry/IP) (ThermoFisher Scientific, Cat#53142) overnight at 4°C prior to its chemical cross-linking reaction182 (link). Dried peptides (1 mg for each sample) were resuspended in 1.4 mL of cold IAP buffer [50 mM MOPS (pH 7.2), 10 mM sodium phosphate and 50 mM NaCl] and centrifuged at maximum speed for 5 min at 4°C to remove any insoluble material. Supernatants (pH ∼7.2) were incubated with the antibody beads for 2 hours at 4°C with gentle end-over-end rotation. After centrifugation at 1000 g for 30 s, beads were washed three times with cold IAP buffer and twice with cold PBS. The diGly peptides were eluted twice with 75 μL 0.15% TFA, desalted using homemade StageTips181 (link) and dried via vacuum centrifugation.
Protein a plus ultralink resin
Manufactured by Thermo Fisher Scientific
Protein A Plus Ultralink resin is a chromatography medium designed for the purification of antibodies. It features high binding capacity and stability, enabling efficient capture and recovery of immunoglobulins from complex samples.
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5 protocols using protein a plus ultralink resin
1
Enrichment of diGly-Modified Peptides
2
Diglycopeptide Enrichment and Profiling
3
diGLY Immunoprecipitation for TMT Proteomics
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diGLY capture was performed largely as described (25 (link)). The diGLY monoclonal antibody (Cell Signaling Technology; D4A7 clone) (32 μg of antibody/1 mg of peptide) was coupled to Protein A Plus Ultralink resin (1:1 μl of slurry/μg of antibody) (Thermo Fisher Scientific) overnight at 4°C before its chemical cross-linking reaction. Dried peptides (1-mg starting material) were resuspended in 1.5 ml of ice-cold immuno affinity purification (IAP) buffer [50 mM Mops (pH 7.2), 10 mM sodium phosphate, and 50 mM NaCl] and centrifuged at maximum speed for 5 min at 4°C to remove any insoluble material. Supernatants (pH ∼ 7.2) were incubated with the antibody beads for 2 hours at 4°C with gentle end-over-end rotation. After centrifugation at 215g for 2 min, beads were washed three more times with ice-cold IAP buffer and twice with ice-cold PBS. The diGLY peptides were eluted twice with 0.15% trifluoroacetic acid, desalted using homemade StageTips, and dried via vacuum centrifugation, before TMT labeling. A detailed protocol describing the diGLY immunoprecipitation for TMT proteomics has been reported (dx.doi.org/10.17504/protocols.io.buyunxww ).
4
ADSC Pretreatment and RNF4 Immunoprecipitation
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ADSCs were pretreated with CSF2RB adenovirus (ADSC-CSF2RB) or NC adenovirus (ADSC-NC) adenoviruses for 2 days. The cells were washed twice with PBS and lysed with cold 1 × lysis buffer (CST #9803) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, 78438) and a phosphatase inhibitor cocktail (AntiProtech, APT008). The lysates were supplemented with 1 mg/mL DTBP (Thermo Fisher Scientific, 20665). The mixtures were then incubated with anti-RNF4 antibody (Proteintech #17810-1-AP) and Protein A Plus UltraLink Resin (Thermo Fisher Scientific, 53142) and rocked overnight at 4 ℃. The protein A beads were washed extensively with lysis buffer. The proteins were eluted from the beads and resolved by IgG elusion buffer (Thermo Fisher Scientific, 1856202). Samples containing a reducing agent (dithiothreitol) were heated and separated by electrophoresis. After being transferred to polyvinylidene fluoride membranes, the proteins were immunoblotted with anti-p-STAT5 (CST #4322) (1/1,000) as described above.
5
Immunoaffinity Enrichment of diGLY Peptides
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diGLY capture was performed largely as described (Rose et al., 2016 (link)) . The diGly monoclonal antibody (Cell Signaling Technology; D4A7 clone) (32 μg antibody/1 mg peptide) was coupled to Protein A Plus Ultralink resin (1:1 μL slurry/ μg antibody) (Thermo Fisher Scientific) overnight at 4°C prior to its chemical cross-linking reaction. Dried peptides (indicated amount in corresponding figures) were resuspended in 1.5 mL of ice-cold IAP buffer [50 mM MOPS (pH 7.2), 10 mM sodium phosphate and 50 mM NaCl] and centrifuged at maximum speed for 5 min at 4°C to remove any insoluble material. Supernatants (pH ∼7.2) were incubated with the antibody beads for 2 hr at 4°C with gentle end-over-end rotation. After centrifugation at 215 × g for 2 min, beads were washed three more times with ice-cold IAP buffer and twice with ice-cold PBS. The diGLY peptides were eluted twice with 0.15% TFA, desalted using homemade StageTips and dried via vacuum centrifugation, prior to TMT labeling.
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