SureLock Tandem Midi Gel Tank

The SureLock Tandem Midi Gel Tank provides for rapid running of midi gels using minimal buffer in a leak-free system. With a set-up time of ~30 seconds, the tank efficiently runs midi gels while also providing consistent performance.

Run conditions for various types of midi gels are provided below. We recommend performing electrophoresis at constant voltage. Note that run-time values are approximate and will vary depending on gel percentage.

Publication-quality protein electrophoresis data is easily generated using the SureLock Tandem Midi Gel Tank and Invitrogen precast midi gels as illustrated in Figure 1, below. In these examples, the same samples were separated on a NuPAGE Bis-Tris 4–12% midi gradient gel and on a Novex Tris-Glycine Plus 4–12% midi gradient gel, using the SureLock Tandem Midi Gel Tank. Both gels produced crisp bands and straight protein lanes. In the NuPAGE gel example, the combination of the gradient gel and the MOPS buffer resolved the protein bands across a broader range of the gel.

Figure 1. Publication-quality protein electrophoresis gel results using NuPAGE Bis-Tris 4-12% and Novex Tris-Glycine Plus 4-12% gradient midi gels and SureLock Tandem Midi Gel Tank. Both gels were loaded as follows: Lanes 1, 20: 5 µL PageRuler Broad Range Unstained Protein Ladder (Cat. No. 26630); lanes 2-7: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg HeLa lysate, respectively; lanes 8, 13: 5 µL Novex Mark12 Unstained Standard (Cat. No. LC5677); lanes 9-12: 240 ng, 180 ng, 120 ng, 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14-19: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg E. coli lysate, respectively. Electrophoresis was conducted with NuPAGE MOPS running buffer, and Novex Tris-glycine running buffer for each corresponding gel. Gels were stained with SimplyBlue Safe Stain.

Publication-quality, multiplexed western blotting data can be generated using the SureLock Tandem Midi Gel Tank, Blot Modules, and Invitrogen precast midi gels as illustrated in Figure 2, below. In these examples, the same samples were separated on NuPAGE Bis-Tris 4–12% midi gradient gels using the SureLock Tandem Midi Gel Tank and transferred using SureLock Tandem Midi Blot Modules. One gel was transferred to a 0.2 µm nitrocellulose membrane and the second to a 0.45 µm PVDF membrane, and both were processed with the same western blotting protocol, resulting in multiplexed blots with crisp, bright bands with straight lanes.

Figure 2. Multiplex western blotting following separation in the SureLock Tandem Midi Gel Tank and wet tank transfer using SureLock Tandem Midi Blot Modules generates excellent results on both nitrocellulose and PVDF membranes. A 2-fold dilution series of A431 cell lysate starting at 1.0 µg/µL was used along with iBright Prestained Protein Ladder. 10 µL of each A431 lysate concentration was loaded in each of 2 NuPAGE 4–12% Bis-Tris, 20-well gels and separated by electrophoresis using the SureLock Tandem Midi Gel Tank with the Invitrogen PowerEase Touch 350 W Power Supply and pre-programmed protocol NuPAGE BT (MOPS) (200 V; 40 min). Proteins were transferred to either a 0.2 µm nitrocellulose membrane or a 0.45 µm PVDF membrane using the SureLock Tandem Midi Blot Module in the SureLock Tandem Midi Gel Tank at 25 V for 30 min. Membranes were processed using the Invitrogen Bandmate™ Automated Western Blot Processor. Membranes were blocked in 1X Clear Milk Blocking Buffer. For fluorescence multiplex detection, the membranes were probed with a mixture of primary antibodies diluted in 1X blocking solution containing rabbit-anti EGFR (1:1,250) (Cat. No. PA1-1110), rabbit-anti Hsp90 (1:4,000) (Cat. No. PA3-013), chicken anti-Calreticulin (1:4,000) (Cat. No. PA1-903), mouse anti-actin (1:1,250) (Cat. No. MA1-140), and mouse anti-p23 (1:1,250) (Cat. No. MA3-414) followed by an incubation with secondary antibodies diluted at 1:5,000 in 1X blocking solution: Goat anti-Mouse Alexa Fluor Plus 488 (Cat. No. A32723), Goat anti-Chicken Alexa Fluor 546 (Cat. No. A11040), and Goat anti-Rabbit Alexa Fluor Plus (Cat. No. A32735). Membranes were imaged on the iBright FL1500 with 3x3 binning, and 1.0X zoom.

The amount of protein one can load onto a protein gel well affects the ability to detect the protein following protein gel electrophoresis or western blotting: the more you can load, the easier it is to detect. The protein load capacities of the three Invitrogen precast midi gel chemistries run in the SureLock Tandem Mid Gel Tank were compared against Bio-Rad Criterion midi gels run in a Bio-Rad Criterion Cell Midi Cell Tank using manufacturer instructions. Decreasing amounts of HEK293 cell lysate prepared in RIPA lysis buffer (48–0.5 µg total protein) were denatured in the respective manufacturer’s sample buffers and subjected to electrophoresis using manufacturer instructions. Table 1 lists the samples, protein mass, and % RIPA buffer loaded in each lane.

In general, Invitrogen gels outperformed Bio-Rad gels at higher lysate loads with Bio-Rad gels suffering from streaking and bowing of bands above 24 µg protein. With immunoblotting, Bio-Rad blots showed band loss and smearing at higher loads for all targets investigated.

Table 1.

Figure 4. Western blots using Novex 4–20% Tris-Glycine Plus midi gels display sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels. A Novex 4–20% Tris-Glycine Plus midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module. In parallel, a Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. Both membranes were analyzed for total protein using the No-Stain Protein Labeling Reagent, followed by chemiluminescent immunodetection of three targets: Vinculin, α-Tubulin, and p23. The Bio-Rad blot shows streaking, bowing of bands above 24 µg protein, and bleed-over into the ladder lane for total protein analysis. With immunoblotting, the Bio-Rad blot shows band loss and smearing at higher loads for all targets.For immunodetection membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For chemiluminescent detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:30,000), Rat anti-α Tubulin (1:15,000), and Mouse-anti p23 (1:60,000) followed by an incubation with secondary antibodies in 1X Blocker FL: Donkey anti-Rabbit HRP (1:5,000), Donkey anti-Rat HRP (1:30,000), and Donkey anti-Mouse HRP (1:240,000) for 1 hour. Membranes were incubated for 5 minutes with SuperSignal West Dura Extended Duration Substrate and imaged for the same amount of time on an iBright Imaging System.

Figure 5. Western blots using NuPAGE 4–12% Bis-Tris midi gels generate sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels. A NuPAGE 4–12% Bis-Tris midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. Both membranes were probed for three targets (Vinculin, α-Tubulin, and p23) using fluorescent immunodetection. Band loss and smearing can be seen at the higher loads for all targets on the Bio-Rad blot, while the blot for the NuPAGE Bis-Tris gel offers superior protein loading capacity above 24 µg and crisp, bright bands. For immunodetection, membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For fluorescence multiplex detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:15,000), Rat anti-α Tubulin (1:5,000), and Mouse-anti p23 (1:5,000) followed by an incubation with secondary antibodies diluted at 1:5,000 in 1X Blocker FL: Donkey anti-Rabbit Alexa Fluor Plus 680, Donkey anti-Rat Alexa Fluor 488, and Donkey anti-Mouse Alexa Fluor Plus 800 for 1 hour. Membranes were imaged on an iBright Imaging System, with the same time and image adjustment settings for each blot.

Figure 6. Western blots using NuPAGE 3–8% Tris-Acetate midi gels generate sharp bands at high protein and RIPA lysis buffer loads while larger proteins fail to transfer from Bio-Rad 7.5% TGX midi gels. A NuPAGE 3–8% Tris-Acetate midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module. The Bio-Rad 7.5% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. For these gels, 10 µL of HiMark Prestained Protein Ladder was used as the protein standard. Both membranes were probed simultaneously for two protein targets (BRCA2 and HDAC1) using chemiluminescent immunodetection. BRCA2 was not detected in the Bio-Rad TGX midi gel, which indicates inefficient transfer of this large protein from the Bio-Rad gel. NuPAGE 3-8% Tris-Acetate precast protein gels were designed for electrophoresis and western blotting of large proteins, and large proteins tend to transfer more efficiently from the lower acrylamide concentration near the top of the 3–8% acrylamide gradient gel. For immunodetection membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For chemiluminescent detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti BRCA2 (1:500) and Rabbit anti-HDAC1 (1:5,000,000) followed by an incubation with secondary antibody in 1X Blocker FL: Donkey anti-Rabbit HRP (1:5,000) for 1 hour. Membranes were incubated for 5 minutes with SuperSignal West Dura Extended Duration Substrate and imaged on an iBright Imaging System for the same amount of time and the same image adjustment settings.