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Products for this protocol

• BMH (bismaleimidohexane)
   
  
• BS3 (bis[sulfosuccinimidyl]suberate)
   
  
• Buffer X (optional; see Troubleshooting)
   
  
  • 20 mM PIPES (pH 7.0)
     
    Salts according to the requirements of the investigated complex The pH of PIPES changes -0.009 units per degree centigrade. The buffer must not contain reagents that can react with the cross-linker employed, e.g., dithiothreitol, ß-mercaptoethanol, Tris, or phosphate.
     
    
• Buffers and solutions for SDS-PAGE (see SDS-PAGE of Proteins)
   
  
• Cross-linking stop solution
  • 1 M dithiothreitol (DTT)
     
    
  • 1 M Tris-Cl (pH 7.8)
     
    
• Dimethylsulfoxide (DMSO) (HPLC grade, if possible), 4°C
   
  
• EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide)
   
  
• Purified protein complex
   
  Purify the protein complex using a method that will keep the complex in its native form. Large quantities of complex can be isolated by recombinant tagging of one of the complex components. The other components assemble on the tagged protein in the cell or cell lysate, and the complex is isolated by the interaction of the tag with a tag-specific solid matrix. Elution conditions must be mild so as not to destroy the complex. Either competition (e.g., using the peptide epitope) or enzymatic cleavage in the linker region between the tag and the complex member is suitable for elution. Salts, detergents, or extreme pH usually result in denaturation of protein complexes and are not advisable for elution.
   
  
• SMCC (succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate)
   
  
• 4X SDS-loading buffer
   
  
  • 200 mM Tris-Cl (pH 6.8)
     
    
  • 8% SDS
     
    
  • 0.4% bromophenol blue
     
    
  • 40% glycerol
     
    
• Acrylamide solution, 2%, prepolymerized (optional, see Step 8)
   
  
• Protein molecular weight marker, prestained
   
  
• Reagents for protein staining (see Step 12)
   
  

• Bio-Spin column (Bio-Rad), MicroSpin G-25 column (APBiotech), or NAP column (Amersham Biosciences) (optional; see Troubleshooting)
   
  
• Electrophoresis equipment (see SDS-PAGE of Proteins)
   
  
• Mass spectrometer capable of identifying proteins (Ion trap, reflector MALDI-TOF, triple quadrupole, QTOF, or QSTAR mass spectrometer)
   
  
• Microcentrifuge tubes
   
  
• Software that calculates peptides from protein sequences (e.g., GPMAW, available at http://welcome.to/gpmaw)
   
  
• Ultrafiltration concentrator (e.g., the Centricon Plus-20; Millipore) (optional; see Troubleshooting)
   
  
• Water bath or heating block, 70°C
   
  
• Water bath, refrigerated, 2°C
   
  

1. Place 13 microcentrifuge tubes on ice. Add 26 µl of protein complex solution to each one.
    
   To distinguish between substrates and the products of the cross-link reaction, include a control to which no cross-linker is added.
    
   
2. Weigh out ~1 mg of each cross-linker on a balance and dissolve in a volume of cold DMSO or H₂O that gives a 10 mM solution, as follows:
    
        EDC: 1.9 mg/ml H₂O
    
        BS3: 5.7 mg/ml H₂O
    
        BMH: 2.8 mg/ml DMSO
    
        SMCC: 3.3 mg/ml DMSO
    
   The cross-linkers hydrolyze rapidly and thus should be handled as quickly as possible. Do not use frozen stocks.
    
   
3. Serially dilute the 10 mM stocks of each cross-linker 10-fold and 100-fold in cold water to 1 mM and 0.1 mM, respectively.
    
   
4. Add 3 µl of each cross-linker solution (0.1 mM, 1 mM, and 10 mM) to one of the tubes containing protein complex (from Step 1) to test the optimal cross-linker concentration.
    
   
5. Incubate the tubes for 1 hour at 2°C on ice.
    
   
6. Stop the reaction by adding 1 µl of cross-linking stop solution. Continue incubation for 10 minutes.
    
   
7. Add 10 µl of 4X SDS-loading buffer to each sample. Heat the samples for 10 minutes at 70°C in a water bath or heating block.
    
   
8. Separate the components of the protein complexes on a low-percentage SDS-PAGE gel. Include protein from the control tube (absent any cross-linker).
    
   The percentage of the SDS-PAGE gel is determined by the size of expected cross-linked products and whether it can still be handled; the gel becomes increasingly fragile with lower percentages of acrylamide. A good compromise is a 6% gel, although this may not be required if small proteins are investigated. To increase handling stability of low-percentage gels, add 1/6 volume of a 2% prepolymerized acrylamide solution. A prestained marker allows electrophoresis to be continued until the designated size reaches the bottom of the gel, allowing a better spread of the slower-migrating species.
    
   
9. Once the best cross-linker and its most suitable concentration have been determined, further optimize the cross-linking reaction conditions by varying the time that the cross-linking reaction is allowed to proceed (10 min to 2 hr) and varying the temperature (2°C to room temperature, at 5°C intervals) (see Step 5).
    
   

10. Add the cross-linker of choice to the protein complex sample. Incubate the mixture under the optimized reaction conditions. Include a control tube containing the protein complex but no cross-linking reagent.
     
    
11. Repeat Steps 6-8.
     
    
12. Visualize the proteins by staining. Identify the bands containing cross-linked proteins by comparison of the cross-linked material to the untreated complex on the SDS-PAGE gel.
     
    
13. Excise the bands of interest from the gel, and analyze them by mass spectrometry.