Pro q diamond stain

GST-RG2-164 and GST-RG2-164 7A were eluted from Glutathione Sepharose 4B resin with elution buffer [50 mM tris-Cl (pH 8) and 30 mM glutathione] and concentrated and exchanged into Hepes-Brij buffer [50 mM Na-Hepes (pH 7.4), 1 mM DTT, 5 mM MgCl₂, and 0.02% Brij-35] with an Amicon Ultra-2 10K NMWL filtration unit (UFC201024). Protein concentrations were then estimated by SDS–polyacrylamide gel electrophoresis (PAGE) with BSA standards followed by InstantBlue Coomassie stain (ab119211). In vitro kinase assays were performed with ~2 μg of substrate, 50 ng of AMPK holoenzyme (EMD Millipore, 14-840), 0.2 mM adenosine triphosphate (ATP) (EMD Millipore, 1191-5GM), and 0.3 mM AMP (EMD Millipore, 118110-5GM) where indicated for 30 min at 30°C. Reactions were terminated by adding LDS buffer, and samples were separated by SDS-PAGE. Phosphorylated proteins were detected with a ProQ diamond stain (Thermo Fisher Scientific, P33302) using a modified protocol (69 ), and total proteins were subsequently detected with InstantBlue Coomassie stain.

The protein concentration of separated IEX peak fractions was adjusted to 0.25 mg/ml and 20 µl of each fraction was mixed with 10 µl of 3× loading dye. An amount of 10 µl of samples were run on SDS–PAGE next to each other twice. One gel was stained with Direct Blue stain (C.B.S. Scientific) and the other gel was stained with Pro-Q Diamond stain (Thermofisher). The stained gels were imaged with Imager Gel Doc XR+ System (Bio-Rad) and the band intensity estimated with Image Lab software (Bio-Rad). The relative intensity of the bands was first estimated for each gel separately (relative to the band in lane 1) and the relative phosphorylation was calculated as the ratio between relative intensity of Direct Blue-stained bands and relative intensity of Pro-Q Diamond-stained bands.

Proteins were separated by electrophoresis on 1D-PAGE 15% polyacrylamide gels. Gels were loaded with an equal volume on each lane. Phosphorylated proteins were detected by Pro-Q^® Diamond stain following the manufacturer’s protocol (Invitrogen, Waltham, MA, USA). In short, the gels were fixed in 5% acetic acid and 50% methanol and incubated with Pro-Q^® Diamond stock solution in 25 mL of staining buffer for 1.5 h. The gel was scanned using a Typhoon 9400 (GE Healthcare, Chicago, IL, USA). Subsequently, total proteins were detected by SYPRO^® Ruby stain. The gels were fixed with 50% methanol and 7% acetic acid and stained with SYPRO^® Ruby stain overnight. Phospho-bands were identified according to the molecular weight, normalized to the total lane individually, and then averaged (n = 5 per group). For positive control groups, the STIM1 inhibitor SKF 96365 (Santa Cruz, Dallas, TX, USA) was applied (5 µM) for 72 h, and the calcineurin inhibitor FK506 (Sigma-Aldrich, St. Louis, MO, USA) was applied (30 μM) for 24 h. The same Pro-Q method was applied to the mice atrial tissue (n = 3 per group).

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)}.

Phosphoprotein staining was performed using the Pro-Q Diamond stain (Invitrogen, Eugene, OR, USA), following the manufacturer’s instructions. The Pro-Q Diamond fluorescent dye is highly specific for phosphorylated serine, threonine, and tyrosine residues on peptides and proteins, and will bind these residues in a sequence-independent manner [17 (link)]. Stained gels were viewed and digitized using an Eagle Eye II gel documentation system (Stratagene, San Diego, CA, USA).

The MPK:WRKY proteins were incubated at approximately 1:10 (w/w) ratio for 30 min as described above (except for the lack of radioactive ATP). After separation by 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).