The iBlot® 7-Minute Blotting System vs. Conventional Semi-Dry and Wet Transfer Systems

A successful western detection experiment relies on a quality transfer of proteins from your gel to the blotting membrane. Accuracy of your results is dependent on the transfer efficiency and reliability of your western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and effort. For convenience and time savings, some researchers have switched to semi-dry blotting, but often at a loss of transfer quality. Although both of these methods are commonly used, neither offers the high quality of transfer combined with speed and convenience of the Invitrogen™ iBlot® 7-Minute Blotting System from Life Technologies.

 

Western Transfer Showdown:
Wet Blot vs. Dry Blot

Did you know you can perform a western protein transfer without buffer in 7 minutes? Watch this playful look at the Western Protein transfer process.

Comparison of the Three Types of Electrotransfer Apparatus

We have compared the iBlot® 7-Minute Blotting System to the conventional semi-dry transfer and wet western transfer systems. Our results demonstrate that the iBlot® dry blotting method provides superior immunodetection sensitivity—that is, transfer quality—compared to either semi-dry or wet transfer methods (Figures 1 and 2). In addition, the iBlot® 7-Minute Blotting System is faster and more efficient than other blotting methods (Table 1).

iBlot vs. semi-dry transfer 
Figure 1. High transfer efficiencies achieved using the iBlot® 7-Minute Blotting System. (A) iBlot® dry transfer to nitrocellulose and (B) semi-dry transfer to nitrocellulose of NuPAGE® Novex® 12% Bis-Tris mini gels. Lanes 1–6: 0.0625 µg, 0.125 µg, 0.25 µg, 0.5 µg, 1.0 µg, and 2.0 µg SW480 human colon cancer cell lysate; lanes 7 and 12: 5 µL SeeBlue® Plus2 Pre-Stained Protein Standard; lanes 8–11: 0.5 µL, 1.0 µL, 2.0 µL, 4.0 µL MagicMark™ XP Western Protein Standard.
iBlot vs. wet (tank) transfer

Figure 2. High transfer quality achieved using the iBlot® 7-Minute Blotting System. (A) iBlot® dry transfer to nitrocellulose and (B) wet (tank) transfer to nitrocellulose of NuPAGE® Novex® 12% Bis-Tris mini gels. Lanes 2–7: 0.0625 µg, 0.125 µg, 0.25 µg, 0.5 µg, 1.0 µg, and 2.0 µg SW480 human colon cancer cell lysate; lanes 1 and 8: 5 µL SeeBlue® Plus2 Pre-stained protein standards; lanes 9–12; 0.5 µL, 1.0 µL, 2.0 µL, 4.0 µL MagicMark® XP Western Protein Standard.


Table 1. Comparison of elapsed time for protein transfer with the iBlot® 7-Minute Blotting System to other blotting methods.

 iBlot® 7-Minute Blotting SystemSemi-Dry
Transfer		Wet/Semi-Wet Transfer
Buffer Preparation0 minutes30 minutes30 minutes
Soaking Gel in Transfer Buffer0 minutes20 minutes0 minutes
Assembling Layers2 minutes10 minutes10 minutes
Transfer7 minutes45–90 minutes1–3 hr
Cleanup0 minutes10 minutes10 minutes
Total Elapsed Time9 minutes1 hr, 55 min–
2 hr, 40 min		1 hr, 50 min–
3 hr, 50 min
Time saved with the iBlot® 7-Minute Blotting System—
1 hr, 45 min–
2 hr, 30 min
1 hr, 40 min–
3 hr, 40 min

How They Work

iBlot® 7-Minute Blotting System
Semi-Dry Transfer
Wet Transfer
Preassembled stacks ready for protein transfer containing electrodes, buffer matrices, and PVDF or NC membraneTransfer stack (both top and bottom) composed of sponge and filter paper, soaked in bufferTransfer stack composed of sponge and  filter paper, soaked in a tank filled with buffer

Comparison of Properties

iBlot® 7-Minute Blotting SystemSemi-Dry TransferWet Transfer
Transfer Buffer Required?  
No100–250 mL, or just enough to construct a bubble-free sandwich1–1.5 L, or enough to fill the transfer tank
Transfer Time  
7 min,
plus 2 min transfer preparation		45–90 min,
plus 70 min preparation and assembly		1 hr–overnight,
plus 50 min preparation and assembly
Transfer Quality  
Reproducible and good transfer quality for proteins between 11 and 220 kDa:*
  • Transfer protocols optimized for immunodetection, e.g., iBlot® system–transferred membranes exhibit higher immunodetection sensitivity, using chromogenic and chemiluminescent procedures
Variable and inefficient transfer quality:
  • Reduced buffer capacity limits transfer time, especially for mid- to large molecular weight proteins
  • Small molecular weight proteins may be transferred through membrane and onto filter paper below
  • Membrane and filter paper MUST be cut to exact gel size, otherwise current will short-circuit around the edge of the gel
Reliable and good transfer quality:
  • Increase temperature during blotting, unless buffer is mixed and cooled during blotting keeps current relatively constant
  • High-current power source is typically required for 1–2 hour transfer
Supplemental Equipment Required  
NoneExternal Power SupplyExternal Power Supply
Post-transfer Requirement  
None
  • Wet-soaking filter paper for clean-up
  • Salt deposits on electrodes require regular maintenance
  • Large amount of hazardous buffer to discard
  • Wet-soaking sponges for clean-up
  • Salt deposits on electrodes require regular maintenance
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