Still using your original iBlot or iBlot 2 device? Upgrade to an iBlot 3 Dry Blotting System Today. This unique design combined with the gel matrix technology of iBlot 3 Transfer Stacks allows the system to generate high field strength and increase the transfer speed. The design of the iBlot 3 Western Blot Transfer System reduces the distance between the electrodes and the integrated power supply.Conventional inert electrodes present in other blotting systems result in oxygen generation, which can result in blotting distortion. The copper anode does not generate oxygen gas as a result of water electrolysis, resulting in increased transfer consistency.This format eliminates the need for premade buffers or soaked filter paper, and minimizes handling that can lead to inconsistent performance. The gel matrix of the Bottom and Top Stack incorporate the appropriate anode and cathode buffers to act as ion reservoirs.The rapid transfer without the need for external power supply or premade buffers is possible due to the following features of the iBlot 3 Western Blot Transfer System: Imaging was completed on the Invitrogen iBright FL1500 Imaging System.Ĭomplete transfer of proteins from the gel to the blotting membrane is accomplished in as few as three minutes. Immunoprocessing was completed on the Invitrogen Bandmate Automated Western Blot Processor. A11040) were prepared in TBST and added to the blots for 2 hours. 37587) and were then probed using primary antibodies raised against EGFR, calreticulin, and p23 (Cat. Blots were then blocked with Thermo Scientific Pierce Clear Milk Blocking Buffer (Cat. 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). MES SDS running buffer was used for gel electrophoresis. Invitrogen iBright Prestained Protein Ladder (2 μL) was loaded into lane 12. A serial dilution of Thermo Scientific HEK293 lysate (lysed in Thermo Scientific RIPA buffer) was loaded onto four Invitrogen Bolt 4–12% Bis Tris plus gels. The iBlot 3 device delivers consistent results. Note: GAM = goat anti-mouse, HRP = horse radish peroxidase, TBST = tris-buffered saline with Tween™ (solution), GAR = goat anti-rabbit. Local background corrected volume per lysate was plotted for each lane. The iBright FL1500 instrument was used for image capture. SuperSignal West Dura chemiluminescent substrate was used for detection. Primary antibody was removed and GAR-HRP secondary antibody (1:175,000 in TBST) was added to blots and incubated for 2 hours. 4EBP1 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Blots were incubated with Pierce Clear Milk Blocking Buffer for 30 minutes. Immunoprocessing was completed using the Bandmate Automated Western Blot Processor. After gel electrophoresis, the proteins were transferred using the method and conditions shown above. Invitrogen iBright FL1500 Imaging System was used for image capture. Thermo Scientific SuperSignal West Dura chemiluminescent substrate was used for detection. Primary antibody was removed and GAM-HRP secondary antibody (1:120,000 in TBST) was added to blots and incubated for 2 hours. Hsp70 primary antibody (1:1,000 in clear milk) was added to blots and incubated for 12 hours at room temperature. Blots were incubated with Thermo Scientific Pierce Clear Milk Blocking Buffer for 30 minutes. Immunoprocessing was completed using the Invitrogen Bandmate Automated Western Blot Processor. The iBlot 3 system demonstrates better transfer efficiency.
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