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Protein Enrichment & Clean Up for Mass Spectrometry
Protein enrichment encompasses numerous techniques to isolate cellular proteins based on their sequence, antibody epitope, post-translational modification (PTM) or active binding site. Protein enrichment is essential for isolating low-abundant proteins and/or reducing sample complexity for proteomic analysis. Enrichment of specific proteins or protein complexes can most easily be accomplished using immunoaffinity techniques such as immunoprecipitation, or through PTM specific affinity binding, such as phosphoprotein enrichment. The novel Thermo Scientific ActivX™ probes and enrichment kits enable the specific targeting of several enzyme classes, including kinases, GTPases, and serine hydrolases.
- Comprehensive—reagents and kits for multiple protein enrichment strategies
- Optimized—all reagents and kits are designed to maximize protein yield and reduce nonspecific binding
- Convenient—easy-to-use spin or magnetic formats enable more rapid enrichment of proteins
- Flexible—kits can be used with either cultured mammalian cells or tissues
- Validated—all products have been fully tested and processed samples have been analyzed using Thermo Scientific Mass Spectrometers.
Choose the right protein enrichment products for your application
 |  |  |  |  | |
|---|---|---|---|---|---|
| Phosphoprotein Enrichment Kit | Streptavidin IP-MS Kit | Protein A/G IP-MS Kit | Kinase Enrichment Kit with ATP Probe | ActivX TAMRA-FP Serine Hydrolase Probe | |
| Typical research goals | Discovery proteomics | Targeted proteomics | Targeted proteomics | Discovery proteomics | Discovery proteomics |
| Target | Phosphorylated proteins | Single or homologous proteins | Single or homologous proteins | ATP binding sites | Active serine hydrolase enzymes |
| Binding/labeling mechanism | Metal chelate affinity to phosphate groups | Biotinylated antibody affinity to target | Antibody affinity to target | Biotinylated ATP analog to active site lysine | TAMRA-labeled fluorophosphonate binds to active site serine |
| Enrichment of target | Pre-digestion (protein) | Pre-digestion (protein) | Pre-digestion (protein) | Post digestion (peptide) | Post digestion (peptide) |
| Enrichment format | IMAC | Immobilized Streptavidin | Immobilized Protein A/G | Immobilized Streptavidin | Immobilized anti-TAMRA mAb |
| Base bead | Agarose | Magnetic | Magnetic | Agarose | Agarose |
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Automated Magnetic Separations for Proteomics
Magnetic beads and KingFisher Purification Systems enable the automation of immunoprecipitation (IP) and other affinity purification protocols for proteomics analysis by mass spectrometry.
Featured data
| Target | A431 | HEK293 | |||
|---|---|---|---|---|---|
| Anti-target Ab | Negative control | Anti-target Ab | Negative control | ||
| % Sequence coverage | EGFR | 65% | 0% | 16% | 0% |
| AKT1 | 36% | 2% | 68% | 6% | |
| AKT2 | 50% | 0% | 82% | 0% | |
| AKT3 | 8% | 0% | 62% | 0% | |
| PTEN | 16% | 0% | 36% | 0% | |
Enrichment of medium- to low-abundant EGFR-AKT pathway targets using Thermo Scientific Pierce Streptavidin Magnetic Beads for MS analysis. EGFR-AKT pathway targets were immunoprecipitated from two cell lines (A431 and HEK293) with biotinylated antibodies and captured with Pierce Streptavidin Magnetic Beads. IP eluates were digested in-solution, and analyzed by LC-MS/MS to assess sequence coverage and identify isoform-specific peptides.
Western blot and MS workflows for targeted capture and analysis of enzymes. The schematic depicts two parallel workflows for the profiling, capture and detection of kinases, GTPases or serine hydrolases with ActivX Probes. Preincubation of enzymes with inhibitors allows for the determination of inhibitor specificity, binding affinity and potency by Western blot (or fluorescent SDS-PAGE for serine hydrolases only) of probe-labeled proteins or MS of probe-labeled peptides.
| Target | A431 | HEK293 | |||
|---|---|---|---|---|---|
| Anti-target Ab | Negative control | Anti-target Ab | Negative control | ||
| % Sequence coverage | EGFR | 65% | 0% | 16% | 0% |
| AKT1 | 36% | 2% | 68% | 6% | |
| AKT2 | 50% | 0% | 82% | 0% | |
| AKT3 | 8% | 0% | 62% | 0% | |
| PTEN | 16% | 0% | 36% | 0% | |
Enrichment of medium- to low-abundant EGFR-AKT pathway targets using Thermo Scientific Pierce Streptavidin Magnetic Beads for MS analysis. EGFR-AKT pathway targets were immunoprecipitated from two cell lines (A431 and HEK293) with biotinylated antibodies and captured with Pierce Streptavidin Magnetic Beads. IP eluates were digested in-solution, and analyzed by LC-MS/MS to assess sequence coverage and identify isoform-specific peptides.
Western blot and MS workflows for targeted capture and analysis of enzymes. The schematic depicts two parallel workflows for the profiling, capture and detection of kinases, GTPases or serine hydrolases with ActivX Probes. Preincubation of enzymes with inhibitors allows for the determination of inhibitor specificity, binding affinity and potency by Western blot (or fluorescent SDS-PAGE for serine hydrolases only) of probe-labeled proteins or MS of probe-labeled peptides.
Additional protein enrichment mass spec products
Resources
For Research Use Only. Not for use in diagnostic procedures.