Transfected cells from the indicated number of 10-cm plates were lysed in 1ml per 10-cm plate of immunoprecipitation buffer (58 (link)) containing protease and phosphatase inhibitors (50mM HEPES pH 7.4, 100mM NaCl, 50mM NaF, 10mM NaH2PO4, 2mM EDTA, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 0.5μM microcystin, 1X Roche cOmplete EDTA-free Protease Inhibitors), aliquots of pre-cleared lysates were taken for loading controls, and the indicated proteins were immunoprecipitated with 2μl of rabbit 6325 antiserum (10 (link)) per 10-cm plate for GC-A proteins or 2μl of rabbit 6327 antiserum (11 (link)) per 10-cm plate for GC-B proteins and 25μl of a 50% slurry of Protein-A-agarose beads (Repligen) per 10-cm plate overnight. The beads were washed three times with immunoprecipitation buffer lacking NaCl. After boiling for 5 minutes in 2X reducing sample buffer, samples were fractionated on 8% resolving gels. The gel was first stained with ProQ-Diamond (Molecular Probes) and then stained with SYPRO Ruby protein stain (Molecular Probes) as previously described (38 (link)). Lysates used for full length GC-A or GC-B western blots were fractionated on 8% resolving gels, and lysates used for β-actin or soluble GC-A intracellular domains were fractionated on 10% resolving gels.
Pro q diamond
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pro-Q Diamond is a fluorescent stain for the detection of phosphoproteins in polyacrylamide gels. It is designed to selectively stain phosphate groups attached to serine, threonine, and tyrosine residues in proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sypro ruby, by Thermo Fisher Scientific (8 mentions)
Pro q diamond, by Bio-Rad (4 mentions)
Chemidoc, by Bio-Rad (3 mentions)
Phos tag, by Fujifilm (2 mentions)
Typhoon 9400 scanner, by GE Healthcare (2 mentions)
Molecular imager fx, by Bio-Rad (2 mentions)
His spintrap, by GE Healthcare (2 mentions)
Anti phospho ser antibody, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions)
Intercept pbs blocking buffer, by LI COR (2 mentions)
Anti sheep irdye 680rd anti rabbit 800cw, by LI COR (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Protein a agarose beads, by Repligen (1 mentions)
Sypro ruby protein stain, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Gt x700, by Epson (1 mentions)
Fluorophorestar 3000, by Anatech (1 mentions)
39 protocols using pro q diamond
1
Immunoprecipitation and Phosphorylation Analysis of Guanylate Cyclase Proteins
2
Phosphorylation Analysis of RBM20 Proteins
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FLAG-tagged RBM20 proteins expressed in HEK293T cells or immuno-precipitated and subsequently treated with CiAP and λPP were separated by neutral polyacrylamide gel electrophoresis (NuPAGE, Invitrogen). The NuPAGE gel was stained with Pro-Q Diamond (Molecular Probes) according to the manufacturer’s instruction, and fluorescence images were acquired with FluoroPhore Star3000 (Anatech). The gel was then stained with Gel-Negative Staining Kit (Nacalai). For Phos-tag SDS-PAGE, a 15% polyacrylamide gel without or with 25 µM Phos-tag (Wako) and 50 µM MnCl2 was prepared and layered with a standard 4.5% stacking gel according to the manufacturer’s instruction. Protein samples were run with standard SDS running buffer (Nacalai). Vertical SDS-agarose gel electrophoresis of cardiac proteins from mice were performed essentially as described previously37 (link). The proteins were detected by staining with CBB (Bio Craft). The images of the stained gels were captured with a scanner GT-X700 (Epson) and processed by using Photoshop CC (Adobe).
3
Detecting Phosphorylation and Glycosylation in Proteins
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Detection of phosphoryl groups in proteins was performed according to the method described by [27 (link)]. After SDS-PAGE the proteins in gels were fixed overnight in 50% (v/v) methanol and 10% (v/v) acetic acid. The gel was washed in water and incubated with the fluorescent phosphosensor dye Pro-Q diamond (a phosphoprotein gel stain; Molecular Probes) during 2 h, under slow agitation, in the dark, and subsequently destained in 15% (v/v) propylenoglycol containing 50 mM sodium acetate, pH 4.0. The gel was finally analyzed in a fluorimeter Typhoon 8000 (Amersham; excitation λ = 532 nm; emission λ = 583 nm) and photographed. The same gel was then stained for total protein with Coomassie Brilliant Blue R-250.
Detection of carbohydrate residues in proteins was performed by affinoblotting. Proteins separated by SDS-PAGE and blotted onto a PVDF membrane were probed for the presence of glycopolypeptides essentially by the concanavalin A/peroxidase method developed by [28 (link)], as described before [29 (link)].
Detection of carbohydrate residues in proteins was performed by affinoblotting. Proteins separated by SDS-PAGE and blotted onto a PVDF membrane were probed for the presence of glycopolypeptides essentially by the concanavalin A/peroxidase method developed by [28 (link)], as described before [29 (link)].
4
SDS-PAGE Phosphoprotein Detection
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Purified proteins were run on 12.5% SDS-polyacrylamide gels. To detect phosphorylation on serine or threonine residues the gels were stained with ProQ Diamond (Molecular Probes) according to the manufacturers recommendations.
5
Phosphorylation of Expressed Proteins
6
Kinase Activity Assay for PAK1 and Cdc42
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Full-length human PAK1 and C-terminally truncated Cdc42 (1-178 amino acids) were expressed in Escherichia coli BL21(DE3) and purified as previously described . Kinase activity was measured in buffer (30 mm HEPES, pH 7.4, 5 mm MgCl 2 , 3 mm dithiothreitol) with 4 μm PAK1 and 15 μm Cdc42-GppNHp (non-hydrolyzable GTP analog). The reactions were started by addition of 100 μm ATP, incubated for 30 min at room temperature, and stopped by addition of 20 μL of 5 × SDS-loading buffer and incubation at 95 °C for 5 min. All samples were analyzed by SDS-PAGE and subsequent staining with both Pro-Q Diamond (Molecular Probes, Invitrogen, Darmstadt, Germany) and colloidal Coomassie, as previously described 24, 25
7
Synthesis and Characterization of Modified Carbon Nanotubes and Nanoparticles
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AP-MWCNTs were purchased from Cheap Tubes. COOH-MWCNTs were synthesized by oxidizing AP-MWCNTs in mixed acids.4 (link) Min-U-Sil silica (quartz) was purchased from US Silica (Frederick, MD, USA). Mesoporous silica was generously provided by Dr. Jeffrey Zink, Department of Chemistry and Biochemistry at UCLA. La2O3, Gd2O3, Sm2O3, Yb2O3 nanoparticles were purchased from Nanostructured & Amorphous Materials (Houston, TX, USA). TiO2 and Bi2O3 were provided by Dr. Lutz Madler at the Department of Production Engineering, University of Bremen, Germany. CQ, Sypro Ruby, Pro-Q-Diamond, LAMP-1 antibody and pHrodo were purchased from Life Technologies (Grand Island, NY, USA). 4-Methylumbelliferyl-beta-d -galactopyranoside, 3-methyladenine and Rapa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-LC-3 was purchased from Abcam (Cambridge, MA, USA). Anti-mTOR, anticathepsin D and anti-ASC antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Recombinant human β-galactosidase was purchased from R&D (Minneapolis, MN, USA). The phosphopeptide, LPSSPVpYEDAASFK, was purchased from Apeptide (Shanghai, China). NLRP3–/– or ASC–/– THP-1 cells are prepared from THP-1 cells that transfected with NLRP3 or ASC shRNA.
8
In Vitro Mps1 and ARHGEF17 Phosphorylation
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200 nM of Mps1 and ARHGEF17 or BSA with 200 nM of each were incubated with kinase buffer containing 12.5 mM Tris-Cl, pH 7.5, 35 mM KCl, 1 mM MgCl2, 50 µM EGTA, 100 µM DTT, and 1× phosStop (Roche) at 30°C for 3 h in the absence or presence of ATP/Mg cocktail (0.25 mM ATP; Merck). The reaction was stopped with 20 mM EDTA. The samples were separated by SDS-PAGE gel and stained with colloidal Coomassie (Sigma-Aldrich), or they were blotted and the phosphorylated protein was visualized with Pro-Q Diamond (Life Technologies) and visualized using the Typhoon imaging device (Fuji).
9
Quantitative Kinase Assay for Phosphorylation
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To validate the MS-MS data for phosphorylation sites; quantitative and qualitative in vitro kinase assays were performed with either using ProQ Diamond (Invitrogen) or ADP-Glo™ Kinase Assay (Promega) according to manufacturer’s instruction. For ADP-Glo assay, PknG (25 ng) was used as a kinase and incubated either with EspJ or with the mutants (100 ng) in buffer containing 40 mM Tris (pH 7.5), 2 mM MnCl2, 20 mM MgCl2, 2 mM DTT, 0.5 mg/ml BSA, and 0.1 mM ATP for 1 h at 26 °C. The assay was also standardized using different concentrations of substrate (100, 50 and 25 ng).
10
Phosphoproteome profiling by 2D-gel
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Extracted leaf total proteins were separated by 2-DE and then 2D gels were stained with Pro-Q Diamond (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, the gels were fixed twice for 30 min/each time, and washed three times with ddH2O for 10 min/each time. The gels were incubated in Pro-Q Diamond stain in darkness for 2 h. The gels were destained with 20% acetonitrile in 50 mM sodium acetate (pH 4.0) for four times (30 min/each time), and then thrice washed for 5 min/each time. Finally, the gels were scanned on a TyphoonTM 9400 scanner (GE Healthcare, USA) with a 532 nm excitation laser and a 610 nm long pass filter with a resolution of 100 microns. After fluorescent image acquisition, the gels were stained with CBB to visualize total proteins. In addition, the same 2D gels were stained with CBB to visualize total proteins and used as control. The protein spots stained by Pro-Q Diamond were further identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) according to Lv et al.21 (link).
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