Introduction

Instructions are provided below for blotting Novex® Pre-Cast Gels using the XCell II™ Blot Module. For more information on the XCell II™ Blot Module, refer to the manual (IM-9051).
 
If you are using any other blotting apparatus, follow the manufacturer’s recommendations.

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Materials Needed

Materials supplied by user:

You will need following items:

  • Blotting membranes
  • Filter paper (not needed if using Novex® pre-cut membrane/filter paper sandwiches)
  • Methanol (if using PVDF membranes)
  • XCell II™ Blot Module
  • Appropriate Transfer Buffer
  • Deionized water
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Ordering Information

Sku Name Size Price Qty
LC5310 Novex® IEF Cathode Buffer pH 3-10 (10X) 125 mL USD 89.00
LC5371 Novex® IEF Sample Buffer pH 3-7 (2X) 25 mL USD 19.41
LC5311 Novex® IEF Sample Buffer pH 3-10 (2X) 25 mL USD 18.25
LC5300 Novex® IEF Anode Buffer (50X) 100 mL USD 18.84
LC5370 Novex® IEF Cathode Buffer pH 3-7 (10X) 125 mL USD 89.00

Power Considerations for Blotting

During blotting, the distance traveled (gel thickness) between the electrodes is much lower than during electrophoresis requiring lower voltage and lower field strength (volts/distance). However, the cross sectional area of current flow is much greater requiring higher current.
 
Blotting power requirements depend on field strength (electrode size) and conductivity of transfer buffer. The higher the field strength and conductivity of the buffer, the higher is the current requirement (the current decreases during the run as the ions in the buffer polarize). It is important to use a power supply capable of accommodating the initial high current requirement.
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Protocols

Preparing Transfer Buffer
 
For blotting Tris-Glycine, Tricine, and IEF Gels, we recommend using the Tris-Glycine Transfer Buffer.
 
Prepare 1000 ml of 1X Tris-Glycine Transfer Buffer using the Tris-Glycine Transfer Buffer (25X) as follows:

Tris-Glycine Transfer Buffer (25X)                 40 ml
Methanol                                                   200 ml
Deionized Water                                          760 ml
Total Volume                                              1000 ml
 
 
Alternate Transfer Buffers


The Tris-Glycine Transfer Buffer will interfere with protein sequencing. If you are performing protein sequencing, use the NuPAGE® Transfer Buffer or the 0.5X TBE Running Buffer to perform blotting. The NuPAGE® Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.
 
Preparing Blotting Pads

Use about 700 ml of 1X Transfer Buffer to soak the pads until saturated. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed.
 
Preparing Transfer Membrane and Filter

Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwiches.

  • PVDF membrane—Pre-wet PVDF membrane for 30 seconds in methanol, ethanol, or isopropanol. Briefly rinse in deionized water, then place in a shallow dish with 50 ml of 1X Transfer Buffer for several minutes.
  • Nitrocellulose—Place the membrane directly into a shallow dish containing 50 ml of 1X Transfer Buffer for several minutes.
  • Filter paper—Soak the filter paper briefly in 1X Transfer Buffer immediately prior to use.
  •  Gel—Use the gel immediately following the run. Do not soak the gel in transfer buffer.


Western Transfer Using the XCell II™ Blot Module

Wear gloves while performing the blotting procedure to prevent contamination of gels and membranes, and exposure to irritants commonly used in electrotransfer
 
Transferring One Gel

  1. After opening the gel cassette, remove wells with the Gel Knife.

  2. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the “foot” of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller.

  3. Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface.

  4. Use the Gel Knife to push the foot out of the slot in the plate and the gel will fall off.

  5. When the gel is on a flat surface, cut the “foot” off the gel with the gel knife.

  6. Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane on the gel, ensuring all air bubbles have been removed.

  7.  Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles.

  8.  Place two soaked blotting pads into the cathode (-) core of the blot module. The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on pad in the same sequence, such that the gel is closest to the cathode plate.

  9. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components.

  10. Position the gel membrane sandwich and blotting pads in the cathode core of the XCell II™ Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place.

  11. Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber.

  12. Place the gel tension wedge so that its vertical face is against the blot module. Lock the gel tension wedge by pulling the lever forward.

  13. Fill the blot module with 1X Transfer Buffer until the gel/membrane sandwich is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat.

  14. Fill the outer buffer chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run.

  15. Place the lid on top of the unit.

  16. With the power turned off, plug the red and black leads into the power supply. Refer to Recommended Transfer Conditions for transfer conditions.

 
Transferring Two Gels in One Blot Module

  1. Repeat Steps 1–6 above twice to make two gel-membrane assemblies.

  2. Place two pre-soaked pads on cathode shell of blot module. Place first gel-membrane assembly on pads in correct orientation, so gel is nearest cathode plate. (See Diagram 2).

  3. Add another pre-soaked blotting pad on top of first membrane assembly.

  4. Position second gel-membrane assembly on top of blotting pad in the correct orientation so that the gel is nearest the cathode side.

  5. Proceed with steps 8–13 from Transferring One Gel.

  6. Refer to Recommended Transfer Conditions for transfer conditions.










Recommended Transfer Conditions


The transfer conditions for Novex® Pre-Cast Gels using the XCell II™ Blot Module are listed in the table below.
 
Note:   The expected current listed in the table is for transferring one gel. If you are transferring two gels in the blot module, the expected current will double.

Gel
Transfer Buffer
Membrane
Power Conditions
Tris-Glycine Gel
Tricine Gel
1X Tris-Glycine Transfer Buffer with 20% methanol
Nitrocellulose or PVDF
25 V constant for 1-2 hours
Expected Current
Start: 100 mA
LT025