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Reproducible Laemmli PAGE Performance

Long Shelf Life Gels Designed to Improve Consistency, Speed, and Reproducibility

• Christopher Belisle, Ph.D.
• ,
• Kate Smith

(Page 2 of 3)

Performance

Figure 1. Performance at elevated voltages: Various Precision Plus Protein standards were loaded on either a 10% Mini-Protean TGX gel or a 10% NuPAGE gel and run at 200 V, 300 V, and 400 V. Buffer temperature was measured at each voltage; NuPAGE gels generate excessive heat at 300 V or above, negatively impacting performance, while the Mini-Protean TGX gels maintain lower temperatures at elevated voltages and provide superior performance. Click Image To Enlarge +

Figure 1. Performance at elevated voltages: Various Precision Plus Protein standards were loaded on either a 10% Mini-Protean TGX gel or a 10% NuPAGE gel and run at 200 V, 300 V, and 400 V. Buffer temperature was measured at each voltage; NuPAGE gels generate excessive heat at 300 V or above, negatively impacting performance, while the Mini-Protean TGX gels maintain lower temperatures at elevated voltages and provide superior performance.

TGX gels retain superior Laemmli-like separation characteristics using standard sample and Tris-glycine running buffers. The patented modification of the Laemmli buffer system has reduced hydrolysis of the gel matrix over time, thereby extending shelf life up to 12 months and delivering consistent, high-quality performance.

At 300 V, a typical run can be completed in 15 minutes (compared to 30–45 minutes for other precast and handcast gels). This is because TGX gels can be run at higher voltages than other gels without impacting performance (Figure 1). The subsequent transfer step can be completed in 15 minutes at 150 V (compared to 1 hour to overnight). 

This new gel technology delivers uniform band shape and symmetry—lot-to-lot and intra-lot reproducibility ensure consistency of results.

“I think there’s a pervasive feeling among researchers that their own hand-poured gels have better resolution, but I would argue the opposite,” said Dr. Jensen. “The TGX gels provide a facile solution for producing publication-quality figures.”

Mini-Protean TGX gels demonstrate greater linearity of separation, which results in increased MW estimation accuracy when compared to traditional Laemmli precast gels or NuPAGE Bis-Tris gel systems. A linear regression analysis of estimated MW vs. known MW yields a coefficient of linearity (R²) of greater than 0.98 for TGX gels, vs. an R² of only 0.86 for a representative NuPAGE gel.

Dynamic range of quantitation and resolution are also superior with TGX gel technology. TGX gels exhibit greater lane and band symmetry than traditional Laemmli gels or alternative long shelf life gels, even when overloaded.

Downstream Applications

Figure 2. High transfer efficiency of Mini-Protean TGX precast gels: Broad range protein standards were prepared as 50-fold dilutions in Laemmli sample buffer for precast TGX gels or NuPAGE sample buffer for NuPAGE gels. Serial twofold dilutions of this sample were loaded on the 10% precast gels in a volume of 5 µL. Gels were run using Tris/Glycine/SDS buffer for TGX gels or MOPS buffer for NuPAGE gels. The proteins from the gel were then transferred into nitrocellulose membrane in a Mini-Protean Tetra cell using the Mini Trans-Blot® module at 110 V (constant voltage) for 15 minutes with prechilled Towbin buffer. Protein transfer was evaluated by staining the membrane with SYPRO Ruby blot stain and imaging on the Molecular Imager® VersaDoc™ 4000 instrument with a three-second exposure (A). Proteins remaining in the gel following transfer were visualized by staining with Bio-Safe Coomassie stain and imaging with the Molecular Imager GS-800 densitometer (B). Click Image To Enlarge +

Figure 2. High transfer efficiency of Mini-Protean TGX precast gels: Broad range protein standards were prepared as 50-fold dilutions in Laemmli sample buffer for precast TGX gels or NuPAGE sample buffer for NuPAGE gels. Serial twofold dilutions of this sample were loaded on the 10% precast gels in a volume of 5 µL. Gels were run using Tris/Glycine/SDS buffer for TGX gels or MOPS buffer for NuPAGE gels. The proteins from the gel were then transferred into nitrocellulose membrane in a Mini-Protean Tetra cell using the Mini Trans-Blot® module at 110 V (constant voltage) for 15 minutes with prechilled Towbin buffer. Protein transfer was evaluated by staining the membrane with SYPRO Ruby blot stain and imaging on the Molecular Imager® VersaDoc™ 4000 instrument with a three-second exposure (A). Proteins remaining in the gel following transfer were visualized by staining with Bio-Safe Coomassie stain and imaging with the Molecular Imager GS-800 densitometer (B).

The Mini-Protean TGX gels deliver superior staining with low background.  These gels are compatible with all commonly used stains and mass spectrometry applications. Dr. Jensen remarked that using Bio-Rad’s precast gels prevented user contamination that could potentially interfere with mass spectrometric analysis when using handcast gels.

The Mini-Protean TGX precast gels provide transfer efficiency with both wet/tank and semi-dry transfer systems. The proteins from the gel can be transferred onto a PVDF or nitrocellulose membrane in as little as 15 minutes (Figure 2).