FLEX-ible automated western blot processing

Simply load your primary antibody, secondary antibody, and wash solutions into the device and then walk away. The Invitrogen iBind Flex Western Device automatically performs all immunoblotting steps using sequential lateral flow technology, a simple form of capillary action. In less than 3 hours, the blot is ready for final detection. You can continue to use your existing chromogenic, chemiluminescent, or fluorescent western blotting protocols, along with your choice of primary antibody or secondary antibody conjugates of HRP, AP, or fluorescent dyes.

  • Flexibility—process up to one midi blot, two mini blots, or six vertically cut strips, using the same or different conditions
  • Antibody savings—use up to 80% less primary antibody than with traditional tray-based incubation steps for western blotting
  • Load and go—processes solutions using passive sequential lateral flow technology, with no batteries, lab shakers, trays, or timers required
  • Reproducibility—automated blot processing enables improved consistency among blots

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Flexible formats

The iBind Flex system comes with interchangable wells, which allows you to run multiple membrane formats and even run different primary and secondary antibody conditions in the same device at the same time.

Midi blots

Using the same antibody conditions

Mini blots

Using the same or different antibody conditions

Vertically cut strips

Using the same or different antibody conditions


Watch the iBind Flex demo video

Results are as good as or better than manually processed western blots

Comparison of western blots processed manually vs. with the iBind Flex Western System. Samples containing GST-tagged recombinant proteins were separated on Invitrogen NuPAGE 4-12%, 20-well gels in MOPS SDS running buffer and then transferred to nitrocellulose membranes using the Invitrogen iBlot 2 Dry Blotting System. Blots were probed with identical concentrations of the same pair of primary and secondary antibodies. The primary antibody was rabbit anti-GST diluted 1:500 (8 µL in 4 mL  Invitrogen iBind Flex Solution for the iBind system, 40 µL in 20 mL for manual tray incubation). The secondary antibody was goat anti-rabbit HRP diluted 1:600 (6.7 µL in 4 mL iBind Flex Solution for the iBind system, 33.3 µL in 20 mL for manual tray incubation). For final detection, blots were incubated for 5 minutes in Thermo Scientific SuperSignal West Dura Substrate for visualization with an imaging system.

Lanes 1-5: IKK beta (80 ng, 40 ng, 20 ng, 10 ng, 5 ng)
Lanes 6-10: DDR2 (120 ng, 60 ng, 30 ng, 15 ng, 7.5 ng)
Lanes 11-15: FLT1 (40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng)
Lanes 16-20: HCK (360 ng, 180 ng, 90 ng, 45 ng, 22.5 ng)

ibind flex mini

Better western blot results using less primary antibody. Comparison of mini blots processed manually (probing and washing steps performed in a tray) vs. with the Invitrogen iBind Flex Western Device. Blots were produced by separating samples on Invitrogen Bolt 4-12%, 10-well gels with MES SDS running buffer and then transferred to nitrocellulose membranes using the iBlot 2 Dry Blotting System. Each gel contained 10 lanes loaded with two-fold dilution series of 293 cell extracts (30 µg to 0.06 µg). After immunodetection using the conditions described below, blots were incubated for 5 minutes in Thermo Scientific SuperSignal West Dura Substrate and the signal documented with an imager.

  • MEK2 (45 kDa) – rabbit anti-MEK2 antibody applied at 1:1000 for iBind blot processing (= 2 µL in 2 mL of iBind Solution), and 1:1000 for manual blot processing (= 10 µL in 10 mL buffer).
  • Securin(428 kDa) – rabbit anti-Securin antibody applied at 1:1000 for iBind blot processing (= 2 µL in 2 mL of iBind Solution), and 1:1000 for manual blot processing (= 10 µL in 10 mL buffer).
  • Secondary Antibody (both targets): goat anti-rabbit IgG at 1:600 for iBind blot processing (3.33 µL in 2 mL of iBind Solution), and at 1:1800 for manual blot processing (5.55 µL in 10 mL).

ibind flex

Excellent western blot results with vertically cut strips and fluorescence detection. Comparison of mini blots processed manually (probing and washing steps performed in a tray) vs. with the iBind Flex Western Device. Blots were produced by separating samples on Bolt 4-12%, 10-well gels with MES SDS running buffer and then transferred to nitrocellulose membranes using the iBlot 2 Dry Blotting System and then cutting each into three-lane strips. Final imaging was performed using the Odyssey CLx instrument (LI-COR, Inc.).

Samples and lanes were as follows:

  • Lane 1:  Thermo Scientific PageRuler Prestained NIR Protein Ladder (3 µL)
  • Strip 1:  Phosphorylated Akt cell extract (15 µg, 7.5 µg, 3.75 µg) and Elk-1 Fusion Protein (150 ng, 75 ng, 37.5 ng)
  • Strip 2:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)
  • Strip 3:  Phosphorylated Akt cell extract (15 µg, 7.5 µg, 3.75 µg)
  • Strip 4:  Elk-1 Fusion Protein (150 ng, 75 ng, 37.5 ng)
  • Strip 5:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)
  • Strip 6:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)

Probing and antibody conditions were as follows:

Strip # 1° Ab [1° Ab] Manual [1° Ab] iBind Flex 2° Ab [2° Ab] Manual [2° Ab] Flex
1 Phospho-Akt (red)
Phospho-Elk-1 (green)
1:2000
1:1000
1:400
1:200
IRDye 680LT
IRDye 800CW
1:20,000
1:15,000
1:4000
1:3000
2 SRC (red)
beta Catenin (green)
1:250
1:1000
1:50
1:200
IRDye 680LT
IRDye 800CW
1:20,000
1:15,000
1:4000
1:3000
3 Phospho-Akt 1:2000 1:400 IRDye 680LT 1:20,000 1:4000
4 Phospho-Elk-1 1:1000 1:200 IRDye 800CW 1:15,000 1:3000
5 SRC 1:250 1:50 IRDye 680LT 1:20,000 1:4000
6 beta Catenin 1:250 1:200 IRDye 800CW 1:15,000 1:3000

After completion of western processing, blots were imaged on the Odyssey CLx instrument.

Comparison of western blots processed manually vs. with the iBind Flex Western System. Samples containing GST-tagged recombinant proteins were separated on Invitrogen NuPAGE 4-12%, 20-well gels in MOPS SDS running buffer and then transferred to nitrocellulose membranes using the Invitrogen iBlot 2 Dry Blotting System. Blots were probed with identical concentrations of the same pair of primary and secondary antibodies. The primary antibody was rabbit anti-GST diluted 1:500 (8 µL in 4 mL  Invitrogen iBind Flex Solution for the iBind system, 40 µL in 20 mL for manual tray incubation). The secondary antibody was goat anti-rabbit HRP diluted 1:600 (6.7 µL in 4 mL iBind Flex Solution for the iBind system, 33.3 µL in 20 mL for manual tray incubation). For final detection, blots were incubated for 5 minutes in Thermo Scientific SuperSignal West Dura Substrate for visualization with an imaging system.

Lanes 1-5: IKK beta (80 ng, 40 ng, 20 ng, 10 ng, 5 ng)
Lanes 6-10: DDR2 (120 ng, 60 ng, 30 ng, 15 ng, 7.5 ng)
Lanes 11-15: FLT1 (40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng)
Lanes 16-20: HCK (360 ng, 180 ng, 90 ng, 45 ng, 22.5 ng)

ibind flex mini

Better western blot results using less primary antibody. Comparison of mini blots processed manually (probing and washing steps performed in a tray) vs. with the Invitrogen iBind Flex Western Device. Blots were produced by separating samples on Invitrogen Bolt 4-12%, 10-well gels with MES SDS running buffer and then transferred to nitrocellulose membranes using the iBlot 2 Dry Blotting System. Each gel contained 10 lanes loaded with two-fold dilution series of 293 cell extracts (30 µg to 0.06 µg). After immunodetection using the conditions described below, blots were incubated for 5 minutes in Thermo Scientific SuperSignal West Dura Substrate and the signal documented with an imager.

  • MEK2 (45 kDa) – rabbit anti-MEK2 antibody applied at 1:1000 for iBind blot processing (= 2 µL in 2 mL of iBind Solution), and 1:1000 for manual blot processing (= 10 µL in 10 mL buffer).
  • Securin(428 kDa) – rabbit anti-Securin antibody applied at 1:1000 for iBind blot processing (= 2 µL in 2 mL of iBind Solution), and 1:1000 for manual blot processing (= 10 µL in 10 mL buffer).
  • Secondary Antibody (both targets): goat anti-rabbit IgG at 1:600 for iBind blot processing (3.33 µL in 2 mL of iBind Solution), and at 1:1800 for manual blot processing (5.55 µL in 10 mL).

ibind flex

Excellent western blot results with vertically cut strips and fluorescence detection. Comparison of mini blots processed manually (probing and washing steps performed in a tray) vs. with the iBind Flex Western Device. Blots were produced by separating samples on Bolt 4-12%, 10-well gels with MES SDS running buffer and then transferred to nitrocellulose membranes using the iBlot 2 Dry Blotting System and then cutting each into three-lane strips. Final imaging was performed using the Odyssey CLx instrument (LI-COR, Inc.).

Samples and lanes were as follows:

  • Lane 1:  Thermo Scientific PageRuler Prestained NIR Protein Ladder (3 µL)
  • Strip 1:  Phosphorylated Akt cell extract (15 µg, 7.5 µg, 3.75 µg) and Elk-1 Fusion Protein (150 ng, 75 ng, 37.5 ng)
  • Strip 2:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)
  • Strip 3:  Phosphorylated Akt cell extract (15 µg, 7.5 µg, 3.75 µg)
  • Strip 4:  Elk-1 Fusion Protein (150 ng, 75 ng, 37.5 ng)
  • Strip 5:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)
  • Strip 6:  HeLa cell extract (30 µg, 15 µg, 7.5 µg)

Probing and antibody conditions were as follows:

Strip # 1° Ab [1° Ab] Manual [1° Ab] iBind Flex 2° Ab [2° Ab] Manual [2° Ab] Flex
1 Phospho-Akt (red)
Phospho-Elk-1 (green)
1:2000
1:1000
1:400
1:200
IRDye 680LT
IRDye 800CW
1:20,000
1:15,000
1:4000
1:3000
2 SRC (red)
beta Catenin (green)
1:250
1:1000
1:50
1:200
IRDye 680LT
IRDye 800CW
1:20,000
1:15,000
1:4000
1:3000
3 Phospho-Akt 1:2000 1:400 IRDye 680LT 1:20,000 1:4000
4 Phospho-Elk-1 1:1000 1:200 IRDye 800CW 1:15,000 1:3000
5 SRC 1:250 1:50 IRDye 680LT 1:20,000 1:4000
6 beta Catenin 1:250 1:200 IRDye 800CW 1:15,000 1:3000

After completion of western processing, blots were imaged on the Odyssey CLx instrument.

Get your iBind Flex Western System Starter Kit now
 

Begin using a simpler, more elegant western blotting process. The Invitrogen iBind Flex Western Starter Kit includes a complete iBind Flex Western Device, plus cards and a solution kit, so you can get started right away. Contains the following Invitrogen products:

  • iBind Flex Western Device
  • iBind Flex Cards (1 box of 10)
  • iBind Flex Solution Kit (Note: fluorescence detection requires FD Solution Kit)

Future of westerns: How to tackle your western blot challenges

Description: Watch our webinar on “Automated Protein Detection” to learn about getting reproducible western blot results with little hands-on time and using less primary antibody than traditional immunodetection methods.

Play now