For pull-down experiment, Drosophila S2 cells were cultured for 3 days (10 dishes) and then collected and lysed in immunoprecipitation (IP) buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1% NP-40, 10% Glycerol, 1.5 mm EDTA, 10 mm NaF, 1 mm Na3VO4) with protease inhibitor cocktail (Sigma, St Louis, MO, USA). Cell lysates were pre-cleared using Glutathione sepharose (GE Healthcare, Little chalfont, UK) and then incubated with 200 μg immobilized GST-WW protein, which was expressed in BL21 E. coli and was purified with Glutathione sepharose. GST protein was used as a negative control. Proteins in pull-down samples were separated by SDS-PAGE, and were stained using Colloidal Blue staining (Invitrogen, Carlsbad, CA, USA). Compared with control sample, specific bands were selected for liquid chromatography coupled with tandem MS analysis in Protein Centre, SIBCB.
Colloidal blue staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
Colloidal blue staining is a laboratory technique used to detect and visualize proteins in polyacrylamide gel electrophoresis (PAGE) and Western blotting analyses. The colloidal blue stain binds to proteins, allowing for their detection and quantification.
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Lab products found in correlation
A6455, by Thermo Fisher Scientific (2 mentions)
Ab22897, by Abcam (2 mentions)
Polyvinylidene fluoride membranes, by Waters Corporation (2 mentions)
Protein g beads, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Bl21 de3 competent cells, by New England Biolabs (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Transferrin alexa 594, by Thermo Fisher Scientific (1 mentions)
Dyngo 4a, by Selleck Chemicals (1 mentions)
Recombinant human chymase, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Novex 6 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Gfp trap beads, by Proteintech (1 mentions)
Edta free protease inhibitor, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain for mass spectrometry, by Thermo Fisher Scientific (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
11 protocols using colloidal blue staining
1
GST-WW Pull-down Assay in Drosophila S2 Cells
2
Quantifying Reovirus Binding to Receptors
3
Purification of Histone H4 Variants
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Full-length human histone H4 cDNA was cloned into the pET-32a(+) vector (Novagen, no. 69015-3) between Xho I and BamH I sites. Histone H4 wild-type and two other mutant (H4Y51F and H4Y88F) plasmids were transformed into BL21 (DE3)–competent cells (New England Biolabs), and proteins were expressed with 0.3 mM IPTG (isopropyl-β-d -thiogalactopyranoside) induction at 18°C overnight and purified according to the manufacturer’s protocol (Novagen). Briefly, the cell pellet was lysed with lysis buffer [50 mM tris-Cl (pH 7.5), 200 mM NaCl, 1.0% NP-40, and 0.1% Na-deoxycholate with a protease inhibitor cocktail] on ice for 30 min, and the supernatant was collected by centrifugation at 13,000 rpm for 10 min at 4°C. His-tagged histone H4 proteins were precipitated using Ni-nitrilotriacetic acid resins (Sigma-Aldrich) with rotation at 4°C for 1 hour. The resins were washed three times with lysis buffer and eluted with lysis buffer containing 150 mM imidazole. Histone H4 protein expression was confirmed by colloidal blue staining (Invitrogen) in addition to Western blotting using antibodies against His-tag and H4.
4
Chymase Uptake in Rat Cardiomyocytes
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Adult rat cardiomyocytes were plated on laminin. The medium was replaced with serum-free medium containing either 0.1% DMSO (vehicle control) or 30 μM Dyngo-4a (Selleckchem, MA) and incubated in a 37 °C tissue-culture incubator for 30 minutes. Recombinant human chymase (2.5 μg/ml, Sigma-Aldrich, MO) or transferrin-Alexa 594 (5 μg/ml, Life Technologies, OR) was added to the medium and incubated for an additional 2 hours at 37 °C. The cells were chilled on ice, and washed three times with PBS, then PBS + 0.5M NaCl, and 3 final PBS washes, and then fixed with 4% paraformaldehyde in PBS for 30 minutes. The uptake of Recombinant human chymase was identified with the human chymase antibody (1:50, Abcam ab2377) with the appropriate secondary antibody Alexa Fluor-594 (1:700, Invitrogen). A separate cohort were lysed in RIPA buffer containing Halt protease inhibitor cocktail (PIERCE), and separated on a Novex™ 6% Tris-glycine gel (Invitrogen) under reducing conditions. Proteins were transferred to a PVDF membrane and probed with myosin heavy chain antibody (1:100, DSHB MF-20) and HRP conjugated secondary antibody (GE Healthcare) then developed with Supersignal West Dura Substrate (PIERCE). Equal loading was confirmed by colloidal blue staining (Invitrogen).
5
Protein Interaction Profiling of EAAT3
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GFP-tagged human EAAT3 (GFP-hEAAT3) and mGFP were transiently expressed in HEK293 cells and solubilized in lysis buffer [1% Triton X-100, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 5 mM NaF, 5 mM sodium pyrophosphate, and a protease inhibitor mixture] and subsequently immunoprecipitated with GFP-Trap beads (Chromotek, Planegg-Martinsried, Germany) overnight at 4°C. After four washes with lysis buffer containing 1% Triton X-100 and additional two washes with lysis buffer containing 1% CHAPS, EAAT3/beads and GFP/beads complexes were incubated with rat brain lysates (RBL; 8.65 mg) overnight at 4°C. To specify the interactions with RBL, analogous EAAT3/beads complex was incubated in 1% CHAPS lysis buffer without RBL at 4°C. After six washes, bound proteins were eluted in SDS sample buffer at 95°C for 3 min. Eluted proteins were size fractionated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and visualized by a colloidal blue staining (Invitrogen).
6
Immunoprecipitation and Immunoblotting of KCNQ2
7
Immunoprecipitation of HA-xCPEB4 Protein
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Stage VI oocytes were injected with 4.9 fmols of in vitro transcribed HA-xCPEB4 + 3’UTR (wild type or phospho-mutants) and collected at indicated times. Oocytes were homogenized with 10 μl/oocyte of IP lysis buffer (20 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% NP-40, 1 mM MgCl2 and 100 mM NaCl) supplemented with H1 kinase buffer and EDTA-free protease inhibitors (Sigma-Aldrich). 280 μl of oocyte extract was pre-cleared with 25 μl of Dynabeads protein G (Invitrogen) and then incubated for 2 hr at 4°C with anti-HA antibody covalently cross-linked to Dynabeads protein G. Immunoprecipitates were washed six times with IP lysis buffer and eluted with Laemmli sample buffer by heating at 65°C for 20 min. Eluates were resolved in SDS-PAGE, and analysed by Western blotting, silver staining (Pierce Silver Stain for Mass Spectrometry, Thermo Fisher Scientific) or colloidal blue staining (Invitrogen). Mass spectrometry analysis was performed at the Mass Spectrometry Core Facility at IRB Barcelona, as described previously.
8
Phosphorylation of Bub1 by CDK1-CyclinB1 and Mps1
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Recombinant GST-Bub1 (12 μg) was incubated with 20 units CDK1-CyclinB1 (New England Biolabs), 0,2 μg Mps1 (TTK; Life Technologies) in protein kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35). 500 μM ATP and 125 nM Calyculin A (Cell Signaling) were added and the reactions were incubated at 30 °C for 1 h. Controls were incubated in kinase buffer and Calyculin A only. 6 μg of phosphorylated protein was resolved on NuPAGE Novex 4–12% Bis-Tris protein gels, which were fixed and stained by Colloidal Blue Staining (Thermo Scientific). The band corresponding to GST-Bub1 was cut out and analysed by mass spectrometry.
9
KCNQ2 Protein Interaction Assay
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Rat brain membranes (RBMs) and cell lysates from tsA201 cells transiently expressing KCNQ2/pECFP‐N1 were prepared as described previously (Foster et al. 2012 ). RBMs and cell lysates were solubilized in 1% Triton X‐100 lysis buffer and incubated overnight with a rabbit anti‐KCNQ2 (AB22897, Abcam, Cambridge, UK; 1–2 μg ml−1) or a mouse anti‐KCNQ2 (N26A/23, NeuroMab, Davis, CA, USA; 1–2 μg ml−1), and rabbit anti‐GFP polyclonal antibodies (A6455, Thermo Fisher; 1–2 μl ml−1), respectively. Antigen–antibody complexes were then incubated with protein G beads (GE Healthcare, Uppsala, Sweden) for 4 h at 4°C. After six washes, bound proteins were eluted in SDS sample buffer at 95°C for 3 min. Eluted proteins were size fractionated on SDS‐PAGE gels, and visualized by a colloidal blue staining (Thermo Fisher). For immunoblot with GST‐Kv7.2C, purified proteins were separated on SDS‐PAGE gels and transferred to polyvinylidene fluoride membranes (Waters, Milford, MA, USA), which were then immunostained with anti‐KCNQ2 (N26A/23, 0.22 μg ml−1) antibody.
10
Oxidative Stress Profiling in Preadipocytes
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3T3-L1 WT and Atp7a−/− preadipocytes were cultured in 60-mm dishes and treated with 5 μM LA 10 d. Cells were washed with ice-cold PBS and collected and lysed with RIPA buffer with EDTA-free protease inhibitor cocktail on ice for 1 h and centrifuged at 3,000 g for 15 min. Protein concentration from the obtained lysate was estimated by the BCS assay. A mixture of 10 μg of total protein per well and 4× sample buffer were loaded to Laemmli SDS-PAGE gel and stained with Colloidal Blue staining (Thermo Fisher, LC6025) to confirm the quality of proteins. Protein lysate was further diluted to a concentration of 1 μg/μL and further prepared for the analysis.
Analysis of protein oxidation state was done using modifications to the Pan-Protein Adductomics approach (72 (link)), which combines nanoflow-liquid and overlapping-window data-independent acquisition, high-resolution tandem mass spectrometry. Further details on sample preparations are provided inSI Appendix, Supplementary Methods .
Analysis of protein oxidation state was done using modifications to the Pan-Protein Adductomics approach (72 (link)), which combines nanoflow-liquid and overlapping-window data-independent acquisition, high-resolution tandem mass spectrometry. Further details on sample preparations are provided in
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