PhosPep - Phosphopeptide enrichment kit 50 rxns
PhosPep - Phosphopeptide enrichment kit 50 rxns
£265 / €295
Supplier |
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Genomine |
Cat. no. |
P5010 |
Kit size |
50 |
Links |
Info Protocol Technical note |
Features
- Phospho specific: Highly selective enrichment of phosphopeptides from complex mixtures of enzyme digest of phosporylated proteins
- Sensitive
- Convenient
- Reproducible
- Non-IMAC
Protein phosphorylation is one of the most frequently occurred posttranslational modifications and plays a critical role in cellular regulatory events. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine and tyrosine residues. In fact, phosphorylation of proteins plays a key role in oncogenesis, cell signaling, apoptosis and immune disorders. Despite the importance and widespread occurrence of this modification, profiling of phosphoproteins in cells is still a challenge, due to the low copy of phosphorylated proteins in cell, even when performed on highly purified protein preparations
Mass spectrometry has been shown to be a reliable and routine tool to identify proteins in a high throughput manner. However, the identification of phosphorylation by mass spectrometry is not a trivial matter and, to this day, is not routine. The detection of phosphopeptides by mass spectrometry in a complex mixture, such as a tryptic mass fingerprint, is a rare occurrence. This is thought to be caused by suppression of the ionization of the mainly negatively charged phosphopeptide in the presence of a large excess of nonphosphorylated peptides
This phosphopeptide enrichment kit provides highly selective enrichment of phosphopeptide from non-phosphorylated complex tryptic digest of proteins and facilitates the identification of phosphopeptide by mass spectrometry.
Mass spectrometry has been shown to be a reliable and routine tool to identify proteins in a high throughput manner. However, the identification of phosphorylation by mass spectrometry is not a trivial matter and, to this day, is not routine. The detection of phosphopeptides by mass spectrometry in a complex mixture, such as a tryptic mass fingerprint, is a rare occurrence. This is thought to be caused by suppression of the ionization of the mainly negatively charged phosphopeptide in the presence of a large excess of nonphosphorylated peptides
This phosphopeptide enrichment kit provides highly selective enrichment of phosphopeptide from non-phosphorylated complex tryptic digest of proteins and facilitates the identification of phosphopeptide by mass spectrometry.
Procedure Summary:
1. Selective binding of phosphpeptide
2. Precipitation of phosphopeptide complex
3. Washing phosphopeptide aggregates
4. Resuspension and dissolving of phosphopeptide aggregates
1. Selective binding of phosphpeptide
2. Precipitation of phosphopeptide complex
3. Washing phosphopeptide aggregates
4. Resuspension and dissolving of phosphopeptide aggregates
Results | Yield: From 1μg(45 pmole) trypsin digest of b-casein, single enrichment recover over 95% mono(2062 Da) and tetra(3124 Da) phosphopeptide | Binding assays: binding of greater than 90 pmoles of phosphopeptide per 10μl of A solution is observed in one reaction |
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Solution A | ||
Solution B | ||
Washing solution | ||
Dissolving solution | ||
Phosphopeptide standard | beta casein tryptic digest 10μg |
Identification of phosphopeptide enriched from beta casein trypsin digest by PSD(post source decay) using MALDI-TOF. Panel A represents the MALDI-TOF spectrum of enriched phosphopeptide from beta casein trypsin digest. Blue asterisk represent the enriched phosphopeptides. Rest of the seven spectrums represent the PSD spectrum of enriched phosphopeptide.
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