30-minute Immunodetection Protocol Using the SNAP i.d.^® 2.0 System

The SNAP i.d.^® 2.0 Protein Detection System is the second generation of the SNAP i.d.^® method for detecting immunoreactive proteins on Western blots. The protocols below are a good, basic starting point for using the system; refer to the user manual for variations on this standard protocol, as well as for antibody recovery.

With this unique, vacuum-driven system, reagents are pulled through the membrane, increasing contact between the reagents and the interior of the membrane, without depending on slow diffusion and agitation. This improvement in three-dimensional reagent distribution decreases the length of time required for immunodetection.

While traditional Western blotting required 4 to 24 hours for the blocking, antibody incubation and washing steps, the SNAP i.d.^® 2.0 protocol takes only 30 minutes with no loss of signal intensity or reduction in blot quality. All immunodetection steps after protein transfer to a membrane (i.e., blocking, washing, and primary and secondary antibody incubations) can be performed with the SNAP i.d.^® 2.0 System.

Required Solutions

• Blocking reagent such as non-fat/low fat dry milk (0.5% or less), casein, bovine serum albumin (BSA, B4287) or other commercially available blocking agents such as Bløk^® reagents.
  
• Antibodies (monoclonal and/or polyclonal)
  
• Detection reagents
  
• Wash buffer: Tris- or phosphate-buffered saline solution, pH 7.4, supplemented with TWEEN^® 20 surfactant (TBST or PBST)

Required Equipment and Materials

• Vacuum source: Pump or other uniform vacuum source that can deliver a sustained minimum pressure of 410 millibar (12 in. Hg) and 34 L/min.
  NOTE: WP61 series Chemical Duty Pumps can be used, but may require longer processing times.
  
• One liter or larger vacuum flask with stopper (for waste collection). A Millex®- FA50 filter (or equivalent) is recommended between the vacuum flask and the vacuum source to protect the vacuum source from contamination.
  
• Vacuum tubing to connect vacuum flask to vacuum source
  
• Forceps (XX6200006P)
  
• Membrane blot with transferred proteins

Setup

1. Place the SNAP i.d.^® 2.0 base on a level bench top.
   
2. Attach the vacuum tubing to the back of the system by pushing the coupling insert on the end of the tubing into the quick disconnect fitting at the back of the system base until it clicks.
   
3. Connect the other end of the tubing to a vacuum source. Use a one-liter vacuum flask as a trap and a Millex^®-FA50 filter (SLFA050) to protect the vacuum source from contamination.
   
   NOTE: Any vacuum source that can deliver 410 millibar (12 in. Hg) and 34 L/min is sufficient. If the vacuum source operates at higher than 410 millibar, the SNAP i.d.^® 2.0 system will automatically regulate the vacuum pressure.

SNAP i.d.^® 2.0 Blot Holder Assembly & General Protocol

1. Hold the blot holder by the support layer (blue edges) and wet the membrane layer (white) with distilled water in the wetting tray provided. Place the wetted blot holder on the rolling pad.
   
2. If required, pre-wet the blot in methanol and water, then place it in the center of the blot holder with the protein side down.
   
3. Roll the blot gently to remove air bubbles, then close the blot holder and roll one more time.
   
4. Open the blot holding frame, flip the blot holder so that it is protein side up, then place it inside the frame. A notch in the blot holder ensures correct placement in the frame.
   
5. Close and lock the frame. Add 30 mL of blocking solution. Press the frame down and turn the system knob to apply vacuum. When frame is completely empty, TURN VACUUM OFF.
   
6. Apply 5 mL (for Mini blot) or 10 mL (for Midi blot) of primary antibody across the surface of the blot holder.
   
7. Incubate for 10 minutes at room temperature. Solution will be absorbed into the blot holder and surface may appear dry.
   
8. Press the frame down and apply vacuum.
   Wait 5–8 seconds until the frame is completely empty.
   
9. With vacuum running continuously, add 30 mL of wash buffer. Repeat the washing step 3 more times (total of 4 washes). When frame is completely empty, TURN VACUUM OFF.
   
10. Apply 5 mL (for Mini blot) or 10 mL (for Midi blot) of secondary antibody evenly across the blot holder surface. Incubate for 10 minutes at room temperature with vacuum off. Again, solution will be absorbed into the blot holder and surface may appear dry.
    
11. Press frame down and apply vacuum. Wait 5–8 seconds until frame is completely empty. With vacuum running continuously, add 30 mL of wash buffer. Repeat the washing step 3 more times (total of 4 washes).
    
12. Turn vacuum off and remove blot holder from frame. Remove blot from blot holder and incubate with the appropriate detection reagent as described in the traditional immunodetection protocol.