Protein Phosphorylation

Purification Enrichment




Product # Product Name Function Format Samples
Processed
Input Sample Size Output
Compatibility
P9740 PHOS-Select™ Iron Affinity Gel Agarose Fe+3-chelate phosphopeptide capture Bulk media 5 Captures 4 µM phosphopeptide maximum per mL packed media LC, MALDI-MS, ESI-MS
PP0410 PhosphoProfile I Phosphopeptide Enrichment Kit Silica Ga+3-chelate phosphopeptide capture Prepacked spin-column 24 Captures 25 nM phosphopeptide maximum per spin column LC, MALDI-MS, ESI-MS


PHOS-Select™ Iron Affinity Gel, (P9740)
  • High capacity (at least 4 micromoles of phosphopeptide per ml of resin)
  • Precharged with iron (III) as a stable chelate
  • Provides single-step phosphocompound purification
  • Supplied as an approximate 50% suspension in 50% glycerol buffered at pH 5.0
  • Supplied with technical bulletin providing detailed procedure for use




PhosphoProfile™ I Phosphopeptide Enrichment Kit, (PP0410)

Mass spectrometry (Matrix Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) or Electrospray Ionization (ESI)) of phosphopeptides from tryptic protein digests is a powerful tool for charactering and identification of phosphorylation sites. A combination of low intrinsic abundance, inefficient ionization, and/or signal suppression of most phosphopeptides may limit or even prevent detection, unless the phosphopeptide content is significantly enriched prior to analysis. This kit conveniently includes all materials needed to enrich for phosphopeptides from digested samples in a robust and unbiased manner (see binding comparison). The phosphopeptide capture matrix is a novel Ga+3 chelate silica based on a proprietary nitriloacetic acid (NTA) analog. The silica beads are approximately 20 microns in diameter with a pore size of 1,000 Angstroms. The matrix is packed into spin columns for easy, microscale affinity capture of phosphopeptides (see workflow).


The complete solution kit – 24 samples up to 25 nmoles phosphophopeptide each

  • Mass spec compatible – save time, reduce sample handling and potential loss
  • Proteomics Grade Trypsin - for clean and complete digests
  • PHOS-Select™ Gallium Spin Columns – high capacity, novel Ga+3 silica media for fast, unbiased capture and recovery
  • Controls – validate your process for confidence and reporting
  • Buffers – enzyme reaction, binding, washing and elution optimized formulations for robust performance
  • Consumable equipment included – matched equipment means no risk of sample loss, additional purchases or waste



Workflow highlighting the use of the spin columns for digestion and selective enrichment of phosphopeptides from sample.


Workflow highlighting the use of the spin columns for digestion and selective enrichment of phosphopeptides from sample.


Binding comparison: Selected portions of HPLC chromatograms demonstrating enrichment of phosphopeptides for IMAC technologies.
Standard phosphopeptides representing the three most common sites of phosphorylation phosphoserine, phosphothreonine, and phosphotyrosine were used. The lyophilized solids were first dissolved in water and an approximately equimolar mixture of the peptides was formulated. Each phosphopeptide was added to the BSA digest at a weight ratio of ~1.7% to produce a total phosphopeptide content of ~5% by weight. Quantitation results are given below. Note that competitor A, B, and C technologies were biased in selecting Peptide 2, while Sigma’s technology bound and eluted the peptides in approximately the same ratio as applied to the column.


HPLC Chromatograms comparing phosphopeptide enrichment by IMAC methods
Kit Recovery of phosphopeptide standards Specificity*
1 2 3 Total
Sigma 59% 52% 74% 59% 50%
Competitor A 6% 19% 11% 13% 28%
Competitor B 39% 56% 17% 42% 28%
Competitor C 37% 65% 37% 46% 25%




Additional Products for Phosphoprotein Analysis
Immobilized Antibodies for the Isolation of Phosphospeptides
Controls and Elution Reagents
Resins and Plates

Immobilized Antibodies for the Isolation of Phosphopeptides
Product Name Product #
Monoclonal Anti-Phosphotyrosine-Agarose from mouse, Purified immunoglobulin, PBS suspension, clone PT-66 A1806
Monoclonal Anti-Phosphoserine-Agarose, Purified immunoglobulin, PBS suspension, clone PSR-45 A8076
Monoclonal Anti-Phosphothreonine-Agarose from mouse, Purified immunoglobulin, PBS suspension A7951
Controls and Elution Reagents back to top
Product Name Product #
Phosphotyrosine-BSA Phosphotyrosine Antibody Inhibitor, 2 mg per ml, Buffered aqueous solution, for blotting and ELISA P3967
Phosphoserine-BSA, Phosphoserine Antibody Inhibitor, 2 mg/ml, for blotting and ELISA P3717
Phosphothreonine-BSA, Phosphothreonine Antibody Inhibitor, 2 mg/ml, for blotting and ELISA P3842
Sodium phenyl phosphate dibasic dihydrate, 95%, elutes phosphoproteins from antibody resins P7751
Resins and Plates back to top
Product Name Product #
Avidin-agarose from egg white, Aqueous glycerol suspension A9207
Avidin (Monomeric)-Agarose from egg white, Aqueous glycerol suspension A2036
Streptavidin-Agarose from Streptomyces avidinii, Buffered aqueous suspension S1638
SigmaScreen ExtrAvidin Coated Plates E3027
SigmaScreen Streptavidin 96-well High Capacity Coated Plates S6940
SigmaScreen Streptavidin 384-well High Capacity Coated Plates S8815
Anti-Mouse IgG (whole molecule)-Agarose from goat, IgG fraction of antiserum, Saline suspension A6531
EZviewä Red Protein A Affinity Gel P6486
EZviewä Red Protein G Affinity Gel E3403
Protein L-Agarose from Peptostreptococcus magnus, recombinant, expressed in Escherichia coli P3351
Protein A coated HS 96-well plates, flat-bottomed 96-well plates, clear polystyrene S1938
Protein G coated HS 96-well plates, flat-bottomed 96-well plates, clear polystyrene S2063


Phosphatase Inhibitor Cocktails
Product Number P5726 P2850
Tyrosine Protein Phosphatases and Acid and Alkaline Phosphatases Ser/Thr Protein Phosphatases and Alkaline Phosphatase L-Isozymes
(--)-p-Bromotetramisole Yes
Cantharidin Yes
Imidazole Yes
Microcystin LR Yes
Sodium molybdate Yes
Sodium orthovanadate Yes
Sodium tartrate Yes

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