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Numerous techniques are available for the isolation of cellular proteins based on their post-translational modification (PTM) or active binding site. Protein enrichment is essential for isolating low-abundant proteins or isolating protein classes. Enrichment of specific proteins or protein complexes can most easily be accomplished using target-specific immobilized antibody or PTM specific affinity binding. The novel Thermo Scientific™ ActivX™ probes and enrichment kits enable the specific targeting of several enzyme classes, including kinases, GTPases, and serine hydrolases.
Phospho- protein Enrichment Kit | Glycoprotein Enrichment Kit, ConA | Glycoprotein Enrichment Kit, WGA | Ubiquitin Enrichment Kit | Kinase Enrichment Kit with ATP Probe | ActivX™ TAMRA-FP Serine Hydrolase Probe | |
Target | Phosphorylated proteins | α-linked mannose and terminal glucose residues | N-acetyl glucosamine (GlcNAc) | Polyubiquitin-ated proteins | ATP binding sites | Active serine hydrolase enzymes |
Binding/ labeling mechanism | Metal chelate affinity to phosphate groups | Lectin affinity to sugars | Lectin affinity to sugars | Antibody affinity to target | Biotinylated ATP analog to active-site lysine | TAMRA-labeled fluoro phosphonate binds to active site serine |
Processing time | 2 hr | <1 hr | <1 hr | <45 min | 6.5 hr | 3 hr |
Enrichment format | IMAC-agarose | Immobilized conA-lectin-agarose | Immobilized WGA-lectin-agarose | Immobilized polyubiquitin affinity-agarose | Immobilized strepavidin-agarose | Immobilized anti-TAMRA mAb-agarose |
90003 (10 columns) | 89804 (10 rxn) | 89805 (10 rxn) | 89899 (15 rxn) | 88310 (16 rxn) | 88318 (6.8 µg) |
For downstream mass spectrometry analysis, peptide enrichment and/or fractionation is often necessary to detect peptides from low-abundance proteins and/or for reducing sample complexity for proteomic analysis.
Kit | Phosphoprotein yield (ug)* | Enrichment time (hours) |
---|---|---|
Pierce Phosphoprotein Enrichment Kit | 300 | 1.5 |
Supplier Q kit | 88 | 4.5 |
Supplier I kit | 52** | 3.5 |
Supplier C kit | 160 | 3 |
Supplier E kit | Too dilute to determine | 5 |
*From 2 mg total protein.
**Based on maximum 1 mg load per manufacturer’s protocol.
For Research Use Only. Not for use in diagnostic procedures.