ProteoPrep® 20 Plasma Immunodepletion Kit

The Search is over. Discover! The ProteoPrep® 20 Plasma Immunodepletion Technology

See proteins previously masked
Removal of twenty high abundance proteins reveals the proteins hidden underneath. As a result these previously masked proteins can now be accurately investigated because they are clearly accessible.

Visualize low abundance proteins
Loading limits of current analytical techniques prevent visualization of many low abundance proteins. By removing the majority of the overall protein mass of human plasma, those low abundance proteins are now relatively higher in concentration, and fall within the limits of detection.

Discover accurately and quickly
The convenient spin column format contains a novel antibody media, which display high specificity and increased capacity through the use of conventional antibodies coupled with small recombinant immunoaffinity ligands.

Sigma innovation is leading the way towards better biomarker discovery through the removal of high abundance proteins from human plasma. The ProteoPrep 20 technology reveals proteins previously not seen by removing 97% of the overall protein content of human plasma at 99% efficiency. The technology specifically removes twenty of the top high abundance proteins utilizing conventional antibodies coupled with small recombinant immunoaffinity ligands. The ProteoPrep 20 technology is advancing the frontier of biomarker discovery.

Frequently Asked Questions

User Protocol (128 Kb PDF)

Technical Article: Protein Depletion for Plasma and Serum Proteomic Analysis (110 Kb PDF)

View the latest posters depicting PROT20 product use:

• Specific Depletion of Twenty High Abundance Proteins from Human Plasma. (212KB)
  
  
• Comparison Of Precipitation Methods Following Depletion Of Twenty High Abundance Proteins From Human Plasma. (204KB)
  
  
• High Specificity Depletion of Twenty High-Abundance Proteins from Human Plasma Facilitates Examination of Plasma Proteome Differences Between Individuals. (270KB pdf)
  

Plasma, no depletion (5.8 µl Plasma, 400 µg)   Plasma has a recognized 2-dimensional electrophoresis pattern. Many of the interesting markers for disease research are masked under the more abundant 20 proteins which make up approximately 99% of the total protein mass.

Competitor Depletion Alternative (67 µl Plasma, 400 µg)   Another popular commercial depletion product somewhat reduces complexity. Much of the 2-dimensional electrophoresis map is still occupied by high abundance proteins that cover many low abundance proteins and prevents loading to visualize the very low copy content.

Mass Spectrometric ID of Proteins Following Depletion using the ProteoPrep 20 Plasma Immunodepletion Kit
Low abundance proteins were identified following PROT20 depletion, which were not visualized from either 400 µg of whole plasma or following depletion of 6 proteins. Thirty-five spots were excised from the 2-DE gel following Coomassie Brilliant Blue staining of the gel using EZBlue™ Gel Stain (Product No. G1041). The protein spots were trypsin digested using the Trypsin Profile In Gel Digestion Kit (Product No. PP0100). The digests from each spot were submitted for MALDI TOF MS. Protein identification was performed using the MASCOT database search algorithm at www.matrixscience.com.		Spot   Protein Identification 1   Alpha-1-B-Glycoprotein 2   Hemopexin (Beta-1B-Glycoprotein) 3   Alpha-2-HS-Glycoprotein 4   Vitamin D Binding Protein 5   Pigment Epithelium-Derived Factor 6   Carboxypeptidase N (Kininase 1) 7   Complement Factor H Fragment 8   Apolipoprotein E 9   Ficolin 3 (Hakata Antigen) 10   PK-120 (Plasma Kallikrein-Sensitive Glycoprotein) 11   Apolipoprotein A-IV 12   Glutathione Peroxidase 3 13   Tetranectin (Plasminogen Binding Protein) 14   Serum Amyloid A (SAA) 15   Kininogen 16   Complement Factor H-related protein 2 17   Inter-alpha-trypsin inhibitor H1 18   Vitronectin 19   Gelatin-Binding Protein 20   Antithrombin III 21   Clusterin (Apolipoprotein J) 22   Inter-alpha-trypsin inhibitor H4

Mass Spectrometric ID of Bound Proteins
Spot   Protein Identification 1   Alpha-1-antichymotrypsin 2   Alpha-1-antitrypsin 3   Immunoglobulin J chain 4   Apolipoprotein CIII 5   Serum Paraoxonase 6   Albumin 7   IgA-2 heavy chain 8   Fibrinogen gamma chain 9   Complement C3 10   Transferrin 11   Cytokeratin 12   Complement C4

Following depletion using the ProteoPrep 20 Plasma Immunodepletion Kit, non-specifically bound proteins were identified.

• Of the spots identified, all but two were fragments of, or related to the 20 specifically depleted proteins.
• Alpha-1-antichymotrypsin is known to have high sequence homology with alpha-1-antitrypsin, one of the 20 depleted proteins.
• Cytokeratin was likely a contaminant during processing.
• Serum Paraoxonase is known to be associated with high density lipoproteins and is likely depleted during the removal of apolipoprotein A-I and A-II.

Bound proteins eluted from the resin were acetone precipitated. 2DE was run on the precipitated protein pellet. Spots (21) were excised from the 2DE gel following Coomassie or silver staining of the gel using the ProteoSilver™ Plus Silver Stain Kit (Product No. PROTSIL2). The protein spots were trypsin digested using the Trypsin Profile In Gel Digestion Kit (Product No. PP0100). The digests from each spot were submitted for MALDI TOF MS/MS analysis. Protein identification was performed using the MASCOT database search algorithm at www.matrixscience.com.

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