Figure 1. ProFound Pull-Down Kit Protocol

• Validated components vs. "homemade" hit-and-miss assemblies Methodologies for identifying or confirming protein:protein interactions are readily available in the literature. Often suggested pull-down assay formulations can introduce several variables that, when applied to your specific system, waste valuable research time that is diverted to troubleshooting resulting anomalies.

• Kits supplied complete with cell lysis buffer
Each kit contains a broad-spectrum cell lysis buffer suitable for bacterial, mammalian and insect cell lines. The buffer is a neutral-pH, Tris-based solution that includes a gentle/patented nonionic detergent formulation that is compatible with the pull-down strategy.

• Flexible protocol aids in the capture of weak or transient interactions
ProFound Pull-Down Kits incorporate a spin column format that eliminates resin loss associated with multiple removals of supernatant from a microcentrifuge tube. Weak interactions have an improved probability of detection when the amount of input resin is constant throughout the assay. In addition, salt concentration and composition are often vital for coaxing weaker protein interactions out of complex protein lysates. ProFound Lysis Buffer Working Solution contains a low-salt concentration of 75 mM NaCl that can be increased to determine the optimal conditions for each unique interaction pair. Similarly, transient interactions can be assayed by addition of co-factors specific to the needs of the interacting pair. Added directly to the lysis buffer, cofactors can tailor binding conditions to "trap" the targeted protein:protein interaction.

• More efficient recovery of interacting complexes
The high-binding capacity of the kit resins allows the use of minimal resin volumes. Reduction in resin volume allows for more efficient elution of interacting complexes. The result is a higher concentration of recovered protein for subsequent gel analysis.

Features and Benefits:

• Provides a complete, affordable and easy-to-use strategy for discovery of protein:protein interactions
• Uses common laboratory equipment (e.g., microcentrifuges and mini-gels)
• Adapts to single- or multiple-sample demands
• Features a flexible pull-down format
• Assays for and detects interactors in one day

Applications:

• Discover a new protein:protein interaction from a cell lysate
• Confirm a putative interaction from a cell lysate or with a previously purified protein
• Extract protein:protein interaction information from in vitro transcription/translation lysates

Kit contents
Each kit contains the appropriate affinity support, a cell lysis buffer, Tris-buffered saline, a suitable eluent, and a supply of spin cup columns and collection tubes to enable you to perform 25 individual pull-down experiments.

Interacting pair model validates the use of Profound Pull-Down Kits for protein interaction discovery
A model system was necessary to develop the use conditions and composition of the components to be provided in kits, enabling the successful performance of a pull-down assay. The system selected to illustrate the effectiveness of the components and protocol in capturing an interaction was that between human protein X-linked inhibitor of apoptosis (XIAP) and the second mitochondrial-derived activator of apoptosis (Smac/DIABLO). The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of apoptosis proteins (IAPs) on caspases. The clone set used in the studies encodes the GST-tagged Smac/DIABLO binding domain of XIAP (referred to as BIR2) and the 9xHis-tagged Smac/DIABLO proteins. A typical pull-down result utilizing either the GST- or PolyHis-tagged formats is illustrated in Figure 2.

Note: Gels were stained with the Pierce GelCode SilverSNAP Stain Kit (Product # 24602).

Acknowledgment
Pierce gratefully acknowledges Dr. Yigong Shi of Princeton University for supplying the GST-BIR2- and 9xHis Smac/DIABlO-expressing clones. Dr Shi's laboratory is engaged in research aimed at understanding the structural and molecular mechanisms involved in tumorigenesis and apoptosis.¹

References

1. Chai, J., Wu, J.-W., Kyin, S., Wang, X. and Shi, Y. (2000). Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406, 855-862.
2. Kaelin, W.G., Jr., Pallas, D.D., DeCaprio, J.A., Kaye, F.J. and Livingston, D.M. (1991). Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64, 521-532. (Discusses the GST pull-down assay)
3. Soutoglou, E., Katrakili, N. and Talianidis, I. (2000). Acetylation regulates transcription factor activity at multiple levels. Mol. Cell 5, 745-751. (Discusses the polyHis pull-down assay)
4. Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual 3rd Edition. Chapter 18: Protein Interaction Technologies, Protocol #3: Detection of Protein-Protein Interactions using the GST Fusion Protein Pull-Down Technique. Cold Spring Harbor Laboratory Press: Plainview, NY.

Ordering Information

Instruction Book   MSDS

Buy Product # Description Pkg. Size Files Price Add 21516 ProFound Pull-Down GST Protein:Protein Interaction Kit Includes: Immobilized Glutathione, 750 µl settled gel ProFound Lysis Buffer, 250 ml Glutathione, 1 gm BupH Tris Buffered Saline, 1 pack (makes 500 ml) Handee Mini-Spin Columns Accessory Pack, 27 columns with pre-inserted frit and top and bottom caps Collection Tubes and Caps Accessory Pack, 100 graduated 2 ml tubes and plug caps Kit $325.00 Add 21277 ProFound Pull-Down PolyHis Protein:Protein Interaction Kit Includes: Immobilized Cobalt Chelate, 750 µl settled gel ProFound Lysis Buffer, 250 ml 4 M Imidazole Stock Solution, 5 ml BupH Tris Buffered Saline, 1 pack (makes 500 ml) Handee Mini-Spin Columns Accessory Pack, 27 columns with pre-inserted frit and top and bottom caps Collection Tubes and Caps Accessory Pack, 100 graduated 2 ml tubes and plug caps Kit $325.00