proq diamond phosphoprotein gel stain  (Thermo Fisher)

Journal: bioRxiv

Article Title: Anti-cancer drug Tamoxifen interferes with Mycobacterium tuberculosis PhoPR mediated signaling and inhibits mycobacterial growth

doi: 10.1101/2023.12.19.571805

Figure Lengend Snippet: In vitro phosphotransfer assay of autophosphorylated sensor kinase PhoR with (A) DevR RR. PhoR was autophosphorylated for 1 h in the presence of γ- 32 P labeled ATP and phosphotransfer reactions to DevR RR were performed for different time points as indicated. (B) Densitometric analysis of the kinetics of DevR phosphorylation by PhoR using the Image J software. Mean ± SD from three independent replicates are plotted. In vitro kinase assays of PhoR with (C) STPK PknK. Both PknK and PhoR kinases were incubated together in the presence of ATP and phosphorylation was followed for different time points as indicated. Visualization was done using ProQ™ Diamond phosphoprotein stain. Top, Autoradiogram or ProQ stained gel; bottom, CBB stained image of the same gel. M, protein marker. (D) Densitometric analysis of the kinetics of PhoR phosphorylation by PknK using the Image J software. Mean ± SD from three independent replicates is presented.

Article Snippet: Ser/Thr phosphorylation was visualized using the ProQ™ Diamond Phosphoprotein Gel Stain (Thermo Scientific™) as per manufacturer’s instructions.

Techniques: In Vitro, Labeling, Software, Incubation, Staining, Marker

Journal: BioMed Research International

Article Title: The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice

doi: 10.1155/2015/742536

Figure Lengend Snippet: Assessment of protein phosphorylation in LV and RV of R21C +/+ mice compared with LV and RV of WT mice. (a) Representative ProQ/Coomassie gels of myofibrillar preparations from LV and RV of R21C +/+ mice compared with LV and RV of WT mice. MyBP-C, myosin binding protein C; F/A, F-actin; TnT, troponin T; Tm, tropomyosin; TnI, troponin I, ELC, myosin essential light chain; RLC, myosin regulatory light chain; +P hcTnI std., phosphorylated human cardiac TnI standard; and +P RLC std., phosphorylated human cardiac RLC standard. (b) Representative Western blot of myofibrillar preparations from LV and RV of R21C +/+ mice compared with LV and RV of WT mice. The level of RLC phosphorylation was determined with phosphospecific RLC antibodies (+P-RLC ab) and compared to the total RLC content assessed with a rabbit polyclonal RLC antibody (CT-1 ab) recognizing total RLC protein. −P RLC std., nonphosphorylated RLC and +P RLC std., phosphorylated RLC standard proteins. (c) Quantification of phosphorylated cTnI was assessed by n = 4 independent SDS-PAGE (ProQ/Coomassie gels) analyses of two to three preparations from LV and RV ventricles per group. (d) Quantification of RLC phosphorylation was assessed by n = 6 SDS-PAGE analyses (Western blots and ProQ/Coomassie gels) of two to three LV and RV preparations from WT and R21C +/+ mice. Note no cTnI phosphorylation in R21C +/+ -KI mouse model. There were no differences in cTnI phosphorylation between the LV and RV of WT mice. Note significantly enhanced phosphorylation in the RV-R21C +/+ compared to LV-R21C +/+ ( P = 0.007) and to LV-WT ( P = 0.027). No statistically significant differences in myosin RLC phosphorylation were observed between the LV and RV of WT mice. Errors bars are SEM.

Article Snippet: After separation of the samples on 15% SDS-PAGE ProQ Diamond phosphoprotein gel stain reagent (Invitrogen) was used (as described in the manufacturer's manual) to assess phosphorylation of troponin (TnT, TnI) and myosin RLC.

Techniques: Binding Assay, Western Blot, SDS Page