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 iBlot™ Dry Blotting System.

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 Dry 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 Dry Blotting System is faster and more efficient than other blotting methods (Table 1).

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Figure 1. High transfer efficiencies achieved using the iBlot Dry 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.

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Figure 2. High transfer quality achieved using the iBlot® Dry 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.


Figure 2. High transfer quality achieved using the iBlot® Dry 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.

Figure 2. High transfer quality achieved using the iBlot® Dry 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.

Figure 2. High transfer quality achieved using the iBlot® Dry 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.

Figure 2. High transfer quality achieved using the iBlot® Dry 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 Dry Blotting System to other blotting methods.

  iBlot Dry Blotting System Semi-dry
transfer
Wet or semi-wet transfer
Buffer preparation 0 minutes 30 minutes 30 minutes
Soaking gel in transfer buffer 0 minutes 20 minutes 0 minutes
Assembling layers 2 minutes 10 minutes 10 minutes
Transfer 7 minutes 45–90 minutes 1–3 hr
Cleanup 0 minutes 10 minutes 10 minutes
Total elapsed time 9 minutes 1 hr, 55 min–
2 hr, 40 min
1 hr, 50 min–
3 hr, 50 min
Time saved with the
iBlot Dry Blotting System

1 hr, 45 min–
2 hr, 30 min
1 hr, 40 min–
3 hr, 40 min

Method overview of the three approaches

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

Comparison of properties

  iBlot Dry Blotting System Semi-dry transfer Wet or semi-wet transfer
Transfer buffer required? No 100–250 mL, or just enough to construct a bubble-free sandwich 1–1.5 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 None External power supply External 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