Protein Interaction – Validation

…to make sure your interaction is specific.

In addition to protein interaction screenings but also as a stand-alone service, PROCOMCURE Biotech offers a set of different validation techniques to choose from. These approaches can estimate whether your interaction is specific, its strength of association and possible inhibitions. Choose from the list of techniques and briefly discuss your project below.

 

Affinity Purification Assay

We will co-express both your protein (A) and the hit protein (B) as affinity tag – fusion proteins to enable the interaction of both proteins in living cells. After lysis, the interacting proteins can then be co-purified from the lysate and will be analyzed via SDS-PAGE.

  • Techniken_final_Affinity Purification Assay

  • Techniken_final_Affinity Purification Assay

    Affin Purif Pic 4

     

     

     

     

     

     

     

     

     

     

    Your protein of interest (A) and the hit protein (B) will be co-expressed as fusion proteins with either a HIS-tag or a GST-tag depending on the properties of your proteins. After expression, cells (E.coli) will be lysed and the lysate loaded onto affinity resins specific for the tag chosen. (A) in complex with (B) will bind the resin via the affinity tag while unbound proteins can be removed by washing the column. The eluted protein fraction will then be resolved on a SDS-PAGE for Krypton staining. To verify that the complex of (A) and (B) was purified, the process can be repeated using an affinity tag for (B).

    • intracellular environment provides advantages for protein folding
    • native environment favors complex formation
    • no previous knowledge of complex composition required
    • additional complex forming proteins can be identified

     

    • steric hindrance from affinity tag
    • false-positive hits from host organism
    • for strong and moderate interactions only

     

    • if the Affinity Purification Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Golemis, Erica (2002). Protein–protein interactions: a molecular cloning manual.
      Plainview, N.Y: Cold Spring Harbor Laboratory Press. ISBN 0-87969-628-1
    • Schon, Eric A.; Pon, Liza A. (2001). Mitochondria.
      Methods in Cell Biology 65. Boston: Academic Press. pp. 218–219. ISBN 0-12-544169-X
    • Stumpf MP, Thorne T, de Silva E, Stewart R, An HJ, Lappe M, Wiuf C (May 2008). “Estimating the size of the human interactome”.
      Proc. Natl. Acad. Sci. U.S.A. 105 (19): 6959–64. doi:10.1073/pnas.0708078105. PMC 2383957. PMID 18474861

     

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FRET Assay

We will co-express your protein (A) and the hit protein (B) as fluorophore – fusion proteins (CFP- and YFP-fusions respectively). A protein-protein interaction will bring both fluorophores in close proximity and thereby enable FRET (fluorescence resonance energy transfer). This leads to an excitation of YFP and the specific YFP-emission can be measured.

  • Techniken_final_FRET2

  • Techniken_final_FRET2

    Fret example 4

    Your protein (A) and the hit protein (B) will be expressed as fluorophore-fusion proteins; (A) as CFP fusion and (B) as YFP fusion. Lysates of both candidates will then be co-incubated and FRET measurements including full spectra in 1 nm steps will be performed. If the protein (A) forms a complex with protein (B) under CFP excitation, fluorescence resonance energy transfer (FRET) will result in YFP emission. Hence no purification steps are required and even weak interactions can be identified.

    • highly sensitive even at low quantities
    • concentrations are determined accurately by absorbance
    • advantageous for weak interactions
    • adaptable for multi-well plate format

     

    • steric hindrance from fluorophore
    • photobleaching may occur

     

    • if the FRET Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Sarah F. Martin, et al (2008). Quantitative analysis of multi-protein interactions using FRET: Application to the SUMO pathway.
      Protein Sci. 2008 Apr; 17(4): 777–784. doi: 10.1110/ps.073369608
    • Helms, Volkhard (2008). Fluorescence Resonance Energy Transfer.
      Principles of Computational Cell Biology. Weinheim: Wiley-VCH. p. 202. ISBN 978-3-527-31555-0
    • Truong, Kevin; Ikura, Mitsuhiko (2001). The use of FRET imaging microscopy to detect protein–protein interactions and protein conformational changes in vivo.
      Current Opinion in Structural Biology 11 (5): 573–8. doi:10.1016/S0959-440X(00)00249-9

     

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BRET Assay

Similar to FRET assays, we will fuse the protein of interest (A) with a bioluminescence producing enzyme (luciferase). The hit protein (B) will be expressed as a YFP fusion protein. A protein-protein interaction will bring the luciferase and the fluorophore in close proximity. Comparable to FRET, a YFP emission signal will be the result of a protein-protein interaction.

  • Techniken_final_BRET2

  • Techniken_final_BRET2Bret3

    Your protein of interest (A) will be expressed as a luciferase fusion protein, whereas the hit protein is attached to the YFP fluorophore. Lysates of both expression cultures will then be co-incubated to enable complex formation. If the protein of interest (A) forms a complex with your hit protein (B) supplementation of coelenterazine (luciferase substrate) will trigger bioluminescence resonance energy transfer (BRET) resulting in measurable YFP emission. Hence no purification steps are required, even weak interactions can be identified, and due to the bioluminescent excitation, photobleaching will be reduced to a minimum.

    • highly sensitive even at low quantities
    • concentrations are determined accurately by absorbance
    • advantageous for weak interactions
    • adaptable to multi-well plate format
    • no photobleaching due to bioluminescent excitation

     

    • steric hindrance from fluorophore and luciferase
    • necessity of substrate supplementation

     

    • if the BRET Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Johan Bacart, et al. (2008). Review: The BRET technology and its application to screening assays.
      Biotechnol. J., 3, 311–324 DOI 10.1002/biot.200700222
    • Qiguang Xie, et al. (2012). Bioluminescence Resonance Energy Transfer (BRET) Imaging in Plant Seedlings and Mammalian Cells.
      Methods Mol Biol.; 680: 3–28. doi: 10.1007/978-1-60761-901-7_1

     

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Fluorescence Polarization Assay

We will express and purify your protein (A) as a fluorophore — fusion protein and excite the fluorophore (F) with polarized light. If the purified hit protein (B) interacts with your protein, the increase in mass will decelerate the molecule movement. A second polarization filter will then detect the increase of polarized light in a given amount of time.

  • Techniken_final_Fluorescence Polarization2

  • Techniken_final_Fluorescence Polarization2flupo 1

     

     

     

     

     

     

     

     

     

    Your protein (A) will be expressed as a fluorophore (F) fusion protein with an additional tag for purification. Hit proteins only receive the tag for purification. Your protein (A) is rotating freely in a buffer solution. Once exposed to a polarized light source, the degree of depolarization is measured as a reference. Addition of your hit protein (B) and the interaction with your protein (A) will result in a slower rotating complex (A-B). Thus, a higher amount of polarized light can be measured in a polarization emission. The compatibility of a multi-well plate format of this assay offers the possibility for the IC50 determination and studies on inhibitors of the interaction.

    • suitable for small peptide (<10 kDa) ligands
    • insensitive to inner-filter effects
    • adaptable to multi-well plate format

     

    • steric hindrance from fluorophore
    • occurrence of auto-fluorescence

     

    • if the FP Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Gregorio Weber (1952). Polarization of the Fluorescence of Macromolecules. 1. Theory and experimental Method
      Biochemical Journal, 51, 145–155
    • Wendy A. Lea. (2012). Fluorescence Polarization Assays in Small Molecule Screening
      Expert Opin Drug Discov. 2011 Jan; 6(1): 17–32 doi: 10.1517/17460441.2011.537322
    • Nathan J. Moerke (2009). Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding
      Curr. Protoc. Chem Biol. 1:1-15. DOI: 10.1002/9780470559277.ch090102

     

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Alpha Screen Assay

We will express and purify your protein (A) and the hit protein (B) as affinity tag – fusion proteins. The fusion proteins will then bind Donor and Acceptor beads and a protein-protein interaction will bring both beads in close proximity. A Donor bead excitation of 680 nm will result in the release of singlet oxygen, triggering a signal cascade in the Acceptor bead and the release of an emission signal at 615 nm.

  • Techniken_final_Alpha Screen2

  • Techniken_final_Alpha Screen2alpha1

    Your protein (A) as well as all hit proteins (B) will be expressed as affinity tag fusion proteins. After purification, your protein (A) can be attached to an AlphaScreen Donor Bead and the hit protein (B) to an Acceptor Bead respectively. Both beads will then be co-incubated to enable complex formation. The AlphaScreen Donor Bead is excited at 680 nm and instead of light emission, singlet oxygen is released. If the hit protein (B) binds your protein (A) and the beads come in close proximity, the singlet oxygen is transferred to the AlphaScreen Acceptor Bead resulting in light emission between 520-620 nm respectively (low background fluorescence). The compatibility of a multi-well plate format of this assay offers the possibility for the IC50 determination and studies on inhibitors of the interaction.

    • low background fluorescence
    • reduced steric hindrance
    • robust to buffer changes
    • compatible in multi-well plate format

     

    • sensitive to intense light
    • efficient bead labeling is different for all proteins

     

    • if the AS Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Richard M Eglen. et al. (2008). The Use of AlphaScreen Technology in HTS: Current Status
      Curr Chem Genomics. 2008; 1: 2–10. doi: 10.2174/1875397300801010002

     

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Biolayer Interferometry Assay

We will express and purify your protein (A) and the hit protein (B) as affinity tag fusion proteins. Your protein will then bind a biosensor tip surface and cause a shift in the interference pattern. Binding of (B) to (A) will result in a change of refraction index that can be monitored in real-time.

  • Techniken_final_Biolayer Interferometry Assay

  • Techniken_final_Biolayer Interferometry AssayBLItz Schema 2

     

     

     

     

     

     

     

     

     

    Your protein (A) as well as all hit proteins (B) will be expressed as affinity tag fusion proteins. After the purification, your protein (A) is attached to the surface of the biosensor tip via the affinity tag. This binding causes a shift in the interference pattern when white light is send through the tip. Next, the purified hit protein (B) is added. Binding of (B) to (A) results in an additional layer at the sensor tip and therefore in a change in refraction index. The change can be monitored in real-time to monitor and can be used to determine binding specificity, rates of association, dissociation and protein concentration. To confirm this complex formation, the hit protein (B) will be bound to another tip and the protein (A) will be added subsequently.

    • label-free technique
    • real-time measurements providing detailed rates of association and dissociation
    • crude sample compatibility
    • low sample volume required

     

    • surface related artifacts
    • steric hindrance through protein immobilization

     

    • if the BLI Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Fang, Y. (2007). Label-free Optical Biosensors in Drug Discovery.
      Trends in Biopharmaceutical Industry, 3, 34-38. doi: 10.3389/fphar.2014.00052
    • Abdiche, Y.N. et al. (2008). Determining Kinetics and Affinities of Protein Interactions Using a Parallel Real-time Label-free Biosensor, the Octet.
      Analytical Biochemistry, 377(2), 209-217 doi: 10.1016/j.ab.2008.03.035

     

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Immunoprecipitation Assay

We will express and purify your protein (A) and the hit protein (B). The protein (A) interacts with the hit protein and the complex binds to a specific antibody against (A). As the antibody is immobilized on beads, the complex can be purified and analyzed via SDS-PAGE.

  • Techniken_final_Immunoprecipitation Assay

  • Techniken_final_Immunoprecipitation AssayAffin Purif Pic 4

    Your protein (A) and the hit protein (B) will be over-expressed to reach the detection limit but no additional label or affinity tags are required. After expression, cells will be lysed and incubated with antibody-coated beads for the specific binding of your protein (A). Washing steps remove unbound non-interacting proteins and the A/B complex can be resolved on a SDS-PAGE with Krypton stain. To verify that the complex of (A) and (B) was purified, the process can be repeated using antibody-coated beads specific for your hit protein (B).

    • label-free technique
    • no steric hindrance due to affinity tags
    • crude sample compatibility

     

    • highly specific antibodies are required
    • lack of tags limits additional analysis

     

    • if the Co-IP Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Ohh M. (2007). The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix.
      Mol Cell. ;1(7):959-68.. doi: 10.1016/S1097-2765(00)80096-9
    • Lee C.(2007). Coimmunoprecipitation assay.
      Methods Mol Biol. ;362:401-6. doi: 10.1007/978-1-59745-257-1_31

     

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Pulldown Assay

We will express and purify your protein of interest (A) and the hit protein (B) as affinity tag fusion proteins. Interacting proteins will be analyzed via affinity purification and SDS PAGE.

  • Techniken_final_Pulldown Assay

  • Techniken_final_Pulldown AssayAffin Purif Pic 4

    Your protein (A) and the hit protein (B) will be co-expressed as fusion proteins, the choice of tag (e.g. HIS-tag, GST-tag) depending on the individual properties of your proteins. After expression, cells will be lysed and loaded on beads specific for the tag of your protein (A). (A) in complex (B) will bind the bead via the affinity tag while intrinsic proteins do not bind and can be removed via multiple washing steps. For evaluation of the process, the eluted protein complex A/B will then be resolved on a SDS-PAGE for Krypton staining. To verify that the complex of (A) and (B) was purified, the process can be repeated using the affinity tag of (B).

    • plasma composition provides advantages for protein folding
    • native environment favors complex formation
    • no previous knowledge of complex composition required
    • additional complex forming proteins can be identified

     

    • steric hindrance from affinity tag
    • false-positive hits from host organism
    • for strong and moderate interactions only

     

    • if the Co-IP Assay is included in your Protein-Interaction offer, no further materials are required from you
    • as stand-alone service we simply need the DNA- or protein sequence in FASTA format

     

    • Einarson, M.B. (2001). Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique.
      Nature Methods – 1, 275 – 276 . doi:10.1038/nmeth2204-27
    • Vikis, H.G. and Guan, K.-L. (2004). Glutathione-S-Transferase-Fusion Based Assays for Studying Protein-Protein Interactions.
      Methods in Molecular Biology 261:175-86 doi: 10.1385/1-59259-762-9:175

     

Discuss your validation project...