Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) |
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| Contributor: |
The Laboratory of Tom Kodadek at the University of Texas, Southwestern Medical Center | ||||||||||||||||||||||||||||||||||||
| Overview |
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| Photo-induced protein cross-linking is a powerful new method for the analysis of protein-protein interactions in vitro. A photo-activated metal-ligand complex mediates the one-electron oxidation of tyrosine or tryptophan residues on a protein. The resultant radical intermediates can cross-link to nearby nucleophilic or aromatic residues rapidly and efficiently. Thus, the metal complex is a mediator or catalyst of cross-linking and the cross-link is the result of direct bond formation between the interacting proteins. In many cases, yields of 20-90% can be obtained with reaction times of one second or less. A detailed protocol and some practical considerations are provided below, along with a list of relevant references. | |||||||||||||||||||||||||||||||||||||
| Procedure |
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A. Protein and Buffer Considerations Purify proteins for Photo Crosslinking through any convenient protein isolation method, such as GST pull-down (such as Protocol ID#417), SDS-PAGE (such as Protocol ID#1919) or sucrose density gradient isolation (such as Protocol ID#951). See Hint #1 for additional details. If possible, proteins should be purified into a solution of Phosphate Buffered Saline (PBS). If the purification process results in the protein being solubulized in a solution other than PBS, dialyze the protein against PBS (see Protocol ID#9054 and use PBS as the dialysis buffer). Buffers other than PBS have been employed successfully; however, easily oxidized buffer components, such as Dithiothreitol or β-Mercaptoethanol should not be included in the buffer. PICUP has proven to be useful in the analysis of protein-protein interactions in more complex settings, such as cell lysates. In such settings, commonly used epitope and affinity tags are usually employed to aid in the analysis of the cross-linked products of interest (see Hint #2). If an antibody that recognizes an unknown epitope is employed to follow PICUP reactions, and the protein of interest appears by Western analysis to be consumed without the production of visible cross-linked products, this probably means that the epitope recognized by the antibody was modified in the course of the reaction. B. Light Source and Reaction Considerations The light source used in the contributor's laboratory is a 150-W xenon arc lamp (Oriel, Stamford, CT). The contributor strongly recommends the use of such an intense light source if available. However, a standard flashlight can support the PICUP reaction. Based on comments from those who have used this methodology, the results are highly dependent on the intensity of the light. Light used in the reaction is first filtered through 10 cm of ddH2 and then a 380 nm to 2,500 nm cut on filter (Oriel). The light exposure time is controlled through the use of the timed shutters of a single lens reflex camera that has had both the lens and back cover removed. If one must use a flashlight, a high-intensity MagLite-type flashlight is recommended. Also, longer irradiation times are necessary (5-30 seconds), and the light source must be closer to the reaction tube (5 cm vs. 50 cm for the 150-W xenon arc lamp). The contributor has not optimized use of flashlight-mediated reactions. For some substrates, PICUP is too efficient. In cases where the protein of interest is driven by multiple cross-linking events into a high molecular weight species, histidine can be added to the reaction. The contributor has found that histidine reduces the reaction efficiency in a predictable way (i.e., increasing histidine concentration results in decreasing reaction efficiency). Inhibition with histidine appears to be a more convenient way to inhibit an overly efficient cross-linking reaction than experimenting with different metal and APS concentrations. For some substrates, PICUP is inefficient. In cases where low yields of cross-linked products are obtained, increase the light reaction time. C. Photo-Induced Crosslinking of Proteins 1. Add 10 to 30 μl of Protein Solution #1 to a 1.5 ml microcentrifuge tube. 2. Add 10 to 30 μl of Protein Solution #2 to the microcentrifuge tube and mix gently (see Hint #3). 3. Immediately prior to exposure to light, add (Ru(bpy))3Cl2 to a final concentration of 125 μM and mix gently. 4. Then add APS to a final concentration of 2.5 mM and mix gently. 5. Photolyze the reaction mixture for 0.5 to 5 sec (see Hint #4). 6. Quench the reaction by adding one-quarter the reaction volume of 4X Gel Loading Buffer (final concentration 1X). 7. Samples are incubated for 5 min at 95°C and separated by SDS-Polyarcylamide Gel Electrophoreses (see Protocol ID#455). 8. Analyze cross-linked products through Coomassie staining (see Protocol ID#716), silver staining (see Protocol ID#496, Protocol ID#1211, Protocol ID#346, or Protocol ID#435) or Western blot analysis (see Protocol ID#70, Protocol ID#1146 or Protocol ID#251). Alternatively, the protein of interest can be radiolabeled and the cross-linked products can be visualized by autoradiography (see Protocol ID#9036). |
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| Solutions |
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| BioReagents and Chemicals |
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SDS Tris(2,2'-Bipyridyl)Ruthenium(II)Chloride, Hexahydrate Potassium Phosphate, Monobasic β-Mercaptoethanol Tris Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride Glycerol Bromophenol Blue Xylene Cyanol |
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| Protocol Hints |
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1. There are numerous protocols for the purification of total protein or a specific protein. Specific protocols identified in the procedure section are examples; browse the Working with Protein section of this web site for additional options. 2. S10, myc epitope tags, His6, and biotin affinity tags have been shown to survive the PICUP reaction in functional form (at least with irradiation times on the order of 1 second or less using the 150W lamp). These tags are useful for either the identification or isolation of cross-linked products. In contrast, the hemagglutin (HA) and FLAG tags are oxidized to products not recognized by the cognate antibody and are therefore not useful tags with which to visualize the outcome of oxidative cross-linking reactions. Some other oxidatively labile tags, such as GFP and fluorescein, have also been shown to be unsuitable for this purpose. 3. The concentration of proteins used can vary. To avoid the generation of spurious cross-linked products, limit the concentration of a single protein to no more than 20 μM final concentration. 4. The microcentrifuge tube is placed parallel to the light source (i.e., the light beam shines into the opening in the top of the tube) at a distance of 50 cm. |
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| Citation and/or Web Resources |
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3. Fancy, D.A., Kodadek, T. Chemistry for the analysis of protein-protein interactions: Rapid and efficient cross-linking triggered by long wavelength light. Proc. Natl. Acad. Sci. USA 1999;96:6020-6024. Also see correction, Vol. 97, Issue 3, 1317a-1317a, February 1, 2000. 1. Fancy, D.A., Denison, C., Kim, K., Xie, Y., Holdeman, T., Amini, F. and Kodadek, T. Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes. Chem. and Biol. 2000;7:697-708. 2. Kim, K., Fancy, D.A. and Kodadek, T. Photo-induced protein cross-linking mediated by palladium porphyrins. J. Amer. Chem. Soc. 1999;121:11896-11897. |
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