An in vitro ΔMKD1 kinase assay using MKKs was performed as previously described (Nishihama et al., 2001 (link)). The assay used 200 ng protein. An in-gel kinase assay was performed using 20 µg total proteins as described elsewhere (Nishiuchi et al., 2006 (link)). An immunoprecipitation kinase assay was performed using anti-MPK4 (a gift from Y. Machida, Nagoya University) and anti-MPK6 (Sigma-Aldrich) antibodies as previously described (Nishiuchi et al., 2006 (link)). The phosphorylation sites were determined by LC-MALDI analysis. The phosphorylated MKK1 and MKK5 proteins were digested by chymotrypsin (Roche). The peptides were analysed using a 4800 Plus MALDI TOF/TOFTM Analyzer (AB Sciex). tandem mass spectrometry (MS/MS) data were evaluated by comparing amino acid substitutions and modifications against the NCBI database using the Paragon algorithm (Shilov et al., 2007 (link)) of the ProteinPilot™ v2.0 software (AB Sciex).
4800 plus maldi tof toftm analyzer
Manufactured by AB Sciex
Sourced in United States, Morocco
The 4800 Plus MALDI TOF/TOF Analyzer is a mass spectrometry instrument designed for accurate and sensitive analysis of biomolecules. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology coupled with tandem time-of-flight (TOF/TOF) mass analyzers to provide high-resolution mass measurements and structural information.
Automatically generated - may contain errors
Lab products found in correlation
Chymotrypsin, by Roche (1 mentions)
Anti mpk4, by Merck Group (1 mentions)
Proteinpilot v2, by AB Sciex (1 mentions)
Anti mpk6, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Fujifilm (1 mentions)
Trypsin, by Promega (1 mentions)
Proteinpilot software, by AB Sciex (1 mentions)
Itraq reagent, by AB Sciex (1 mentions)
Pro q diamond phosphoprotein enrichment kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using 4800 plus maldi tof toftm analyzer
1
In vitro ΔMKD1 kinase assay
2
Leu-Lys Peptide Reaction with 4-HNE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Low-salt solutions of Ac-Leu–Lys–Lys–Lys–Gln (0.029 μmol) and (R)−4-HNE (0.132 μmol) were incubated in PBS (0.01 M, pH 7.2) for 1 h at 37 °C. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on the samples by the 4800 Plus MALDI TOF/TOFTM Analyzer (AB SCIEX, USA).
3
2D Gel Electrophoresis and MALDI-TOF/TOF MS Protein Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The method used was essentially as described by Kondo et al.(25 (link)) with some modifications. Preparative 2DE gels were visualized by CBB-stained. The protein spots picking were performed manually, were destained four times, dehydrated twice, and digested with trypsin (Promega, Madison, WI) solution for overnight at 37°C. The peptide solutions were eluted with 45% acetonitrile/0.1% trifluoroacetic acid (TFA) and concentrated. The peptide solutions containing 50% α-cyano-4-hydroxycinnamic acid (Wako Pure Chemical, Osaka, Japan) were spotted on a MALDI plate. Mass analysis was performed with a matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS; 4800 Plus MALDI-TOF/TOFTM Analyzer, AB SCIEX, Framingham, MA). Protein identification was performed with the MS/MS ion search tool in ProteinPilot software (AB SCIEX).
4
Quantitative phosphoproteomics of Arabidopsis MKD1 mutant
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WT and mkd1 mutant plants were grown on MS-0 medium for 16 d. Then, WT and mkd1 mutant plants were treated on MS agar medium containing 1 µM T-2 toxin for 3 h. Roots of WT and mkd1 mutant plants were collected, and phosphoproteins were purified using the Pro-Q® Diamond Phosphoprotein Enrichment Kit (Invitrogen; Asano and Nishiuchi, 2011 (link)). Total proteins and phosphoproteins were stained with SYPRO Ruby Protein Gel Stain and Pro-Q® Diamond Phosphoprotein Gel Stain, respectively. WT and mkd1 mutant proteins (100 µg each) were labelled using the iTRAQ® Reagents according to the manufacturer’s instructions (AB Sciex). Peptides derived from WT and mkd1 mutant were labelled with tags 114 and 117, respectively. The labelled peptides were analysed using a 4800 Plus MALDI TOF/TOFTM Analyzer (AB Sciex). MS/MS data were evaluated by comparing amino acid substitutions and modifications against the NCBI database using the Paragon algorithm (Shilov et al., 2007 (link)) of the ProteinPilot™ v2.0 software (AB Sciex).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!