iBlot 3 Western Blot Transfer System

Maximum Productivity

Now you can perform reliable, high-quality transfers faster than ever—in as few as 3 minutes. You can also save time that would be spent waiting for transfer device availability. Two independently controlled transfer stations allow you to share the device with your coworker while simultaneously running four mini gels or two midi gels with two different programs. In addition, you can reduce method setup time by selecting preprogrammed methods from the user-friendly touchscreen interface. Programs can also be customized to fit your needs.

Better Transfer Efficiency

With the iBlot 3, you can achieve as good or better protein transfer efficiency compared with wet tank transfer and other rapid transfer methods.

The iBlot 3 system demonstrates better transfer efficiency. (A) Hsp70: Invitrogen NuPAGE 4–12% Bis-Tris mini gels were loaded with 20, 15, 10, 5, 2.5, 1.25, 0.625, and 0.312 μg of A431 lysate per lane, respectively. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Immunoprocessing was completed using the Invitrogen Bandmate Automated Western Blot Processor. Blots were incubated with Thermo Scientific Pierce Clear Milk Blocking Buffer for 30 minutes. Hsp70 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Primary antibody was removed and GAM-HRP secondary antibody (1:120,000 in TBST) was added to blots and incubated for 2 hours. Thermo Scientific SuperSignal West Dura chemiluminescent substrate was used for detection. Invitrogen iBright FL1500 Imaging System was used for image capture. Local background corrected volume per lysate was plotted for each lane. (B) 4EBP1: Invitrogen Novex 16% Tricine mini gels were loaded with 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 μg of A431 lysate per lane, respectively. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Immunoprocessing was completed using the Bandmate Automated Western Blot Processor. Blots were incubated with Pierce Clear Milk Blocking Buffer for 30 minutes. 4EBP1 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Primary antibody was removed and GAR-HRP secondary antibody (1:175,000 in TBST) was added to blots and incubated for 2 hours. SuperSignal West Dura chemiluminescent substrate was used for detection. The iBright FL1500 instrument was used for image capture. Local background corrected volume per lysate was plotted for each lane. Note: GAM = goat anti-mouse, HRP = horse radish peroxidase, TBST = tris-buffered saline with Tween™ (solution), GAR = goat anti-rabbit.

Consistent, Reproducible Results

The pre-assembled, single-use consumables and ultra-fast transfer methods eliminate variability—ensuring consistent and repeatable results compared with classical methods. In addition, the all-new robust design with built-in cooling reduces heat buildup, facilitating run-to-run consistency.

The iBlot 3 device delivers consistent results. A serial dilution of Thermo Scientific HEK293 lysate (lysed in Thermo Scientific RIPA buffer) was loaded onto four Invitrgoen Bolt 4–12% Bis Tris plus gels. Protein loads from lane 1–11, left to right (in μg) were: 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.020, and 0.010. Invitrogen iBright Prestained Protein Ladder (2 μL) was loaded into lane 12. MES SDS running buffer was used for gel electrophoresis. Proteins were transferred using the iBlot 3 Western Blot Transfer Device and iBlot 3 Transfer Stacks, Midi, NC using the broad range (30–250 kDa) preprogrammed transfer method (25V, 6 min, low cooling). Blots were then blocked with Thermo Scientific Pierce Clear Milk Blocking Buffer (Cat. No. 37587) and were then probed using primary antibodies raised against EGFR, calreticulin, and p23 (Cat. Nos. PA1-1110, PA1-903, and MA3-414, respectively) in clear milk for 12 hours. Secondary antibodies (AFP-GAR-800 (Cat. No. A32735), AFP-GAM-488 (Cat. No. A32723), AF-GAChk-546 (Cat. No. A11040) were prepared in TBST and added to the blots for 2 hours. Immunoprocessing was completed on the Invitrogen Bandmate Automated Western Blot Processor. Imaging was completed on the Invitrogen iBright FL1500 Imaging System.

Upgrade Program

Are you still using an original iBlot or iBlot 2 device? Learn more about our incentive program to upgrade to an iBlot 3 device.