Pro q diamond phosphoprotein gel stain

Staining of phosphorylated proteins was performed using Pro-Q™ Diamond Phosphoprotein Gel Stain (Invitrogen) following protein separation on SDS-PAGE. The indicated proteins were incubated for 30 min at 30 °C in the kinase buffer containing 25 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 1 mM PMSF, 25 μM ATP. After separation on 10% SDS-PAGE, the gel was fixed with 50% methanol and 10% acetic acid overnight. The gel was washed with water for 30 min and stained with 3x diluted Pro-Q diamond stain (Invitrogen) in the dark for 2 h. The gel was destained four times for 30 min each with 20% acetonitrile, 50 mM sodium-acetate (pH 4.2). The gel was washed again with water for 10 min and was scanned at 400 V using a Typhoon Scanner (GE Healthcare)^{51 (link)}.

cDNA fragments for Kr-h1(aa1-290), Kr-h1^S154A(aa1-290), Kr-h1(aa89-312), Kr-h1^S154A(aa89-312), and Kr-h1(aa291-591) were separately cloned into pGEX-4t-1 vector (GE Healthcare) for overexpression of recombinant GST-tagged proteins in Escherichia coli Rosetta competent cells (Transgen). Cells were lysed by sonication in lysis buffer with 50 mM Tris-HCl pH 7.5 plus 0.1% Triton X-100 and cleared by centrifugation at 8000×g for 30 min at 4°C. GST-fusion proteins were purified by GST resin (Thermo Fisher Scientific) and incubated with PKCα (SignalChem), followed by SDS-PAGE and Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen).

The recombinant Trx-ZmCPK32 protein was expressed in Escherichia coli Rosetta 2 (DE3) pLysS (Novagen) via isopropyl β-D-1-thiogalactopyranoside induction and purified using Ni-NTA His·Bind Resin (Novagen). To examine kinase activity, 0.5 μg of Trx-ZmCPK32 and 2 μg of histone III were added into the reaction mixture containing kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgSO₄, and 1 mM dithiothreitol) in the presence of either 1 mM CaCl₂ or 2 mM egtazic acid (EGTA). All reactions were initiated by adding 100 μM of ATP at 30°C for 30 min and terminated by adding sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The proteins were separated using 12.5% (w/v) SDS-PAGE and stained with Pro-Q Diamond phosphoprotein gel stain (Invitrogen) as previously described [26 (link), 27 (link)] or Coomassie Brilliant Blue. Phosphorylated protein was visualized using a Molecular Imager FX (Bio-Rad) under 532 nm excitation and a 580 nm bandpass emission filter.

To detect overall phosphorylation changes, a 15% SDS-PAGE gel was stained with Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, P33300, Carlsbad, CA) and destained with Pro-Q Diamond Phosphoprotein Gel Distaining Solution (Invitrogen, P33310, Carlsbad, CA). The gel was then stained with Coomassie G-250 Stain (Bio-Rad Inc., 1,610,786, Hercules, CA) to determine total protein levels. Gel images were captured with ChemiDoc MP (Bio-Rad, Inc., 13,036, Hercules, CA) and band densities were determined and analyzed by using ImageLab 6.0.1 software and Microsoft Excel.

A 15 µg aliquot of protein from total O. tauri cell lysates was run on a Novex NuPAGE 4–12% Bis-Tris by SDS–PAGE with PeppermintStick Phosphoprotein Molecular Weight Standards and a Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific). The gel was fixed overnight (50% methanol, 40% ddH₂O, 10% glacial acetic acid), washed in ddH₂O, and stained with Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, now Thermo Fisher Scientific, Loughborough, UK) in the dark at 25 °C following the manufacturer’s instructions. The gel was imaged on a Typhoon TRIO variable mode imager (GE Healthcare, Amersham, UK) at 532 nm excitation/580 nm emission, 450 PMT, and 50 µm resolution. Images were processed using ImageQuant TL software (GE Healthcare, Amersham, UK). The gel was re-used for protein quantification using SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific) following the manufacturer’s instructions and imaged using a UV transilluminator (Ultra-Violet Products Ltd, Cambridge UK). Protein and phosphoprotein bands were quantified using Image Studio Lite v 4.0 (LI-COR Biosciences, Cambridge, UK).

At 4 weeks after surgery, TAC, MI, and sham animals were euthanized by isoflurane and cervical dislocation at ZT15. Hearts were collected, snap-frozen in liquid nitrogen, and stored at –80°C until use. Cardiac myofilaments were isolated by differential centrifugation using the protocol described by Podobed et al. (34 (link)). Actomyosin MgATPase activity in isolated cardiac myofilaments was determined using a modified Carter assay as described previously (19 (link), 34 (link)). Isolated myofilament proteins were separated using 12% SDS-PAGE, and protein phosphorylation levels were quantified using the PRO-Q Diamond phosphoprotein gel stain (Invitrogen) by following the protocol of Podobed et al. (34 (link)). Gels were then stained with Coomassie to determine total protein. Gel imaging was performed on a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad) and analyzed using ImageJ (NIH) with protein phosphorylation normalized to total protein. Samples were run, stained, imaged at the same time on separate gels, and normalized to actin; see complete unedited blots in the supplemental material.