NuPAGE™ Transfer Buffer (20X), 125mL - FAQs

我们建议丢弃缓冲液，并在再次检查试剂和水的纯度后重新配制。不建议用酸或碱调节pH，因为这会使缓冲液的电导率升高，导致转印期间电流过高。

氯丁醇可用作NuPAGE转膜缓冲液的防腐剂，但对于蛋白质的有效转印并不是必要的。若配制缓冲液时未加入氯丁醇，则应注意缓冲液不能长期稳定保存。建议在配制后2周内用完。

在1块胶和2块胶转膜时，建议在NuPAGE转膜缓冲液中分别加入10%和20%甲醇。

是的，我们推荐在NuPAGE转膜缓冲液中加入NuPAGE抗氧化剂，从而改善还原型蛋白质的转印结果，以便在电泳过程中维持蛋白质的还原状态。

可以，NuPAGE转膜缓冲液可防止氨基酸侧链发生修饰，可兼容通过Edman降解进行的蛋白质N端测序。

DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

Precipitation of the LDS or SDS at 4 degrees C is normal. Bring the buffer to room temperature and mix until the LDS/SDS goes into solution. If you do not want to wait for it to dissolve, you can store the sample buffer at room temperature.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

While they are both Bis-Tris based gels, the chemistries are very different since Bolt gels are optimized for western blotting. Another key difference is the wedge well design of the Bolt gels, which allows larger sample volumes to be loaded.

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The neutral operating pH of the NuPAGE Gels and buffers provides following advantages over the Laemmli system:
-Longer shelf life of 8-12 months due to improved gel stability
-Improved protein stability during electrophoresis at neutral pH resulting in sharper band resolution and accurate results (Moos et al, 1998)
-Complete reduction of disulfides under mild heating conditions (70 degrees C for 10 min) and absence of cleavage of asp-pro bonds using the NuPAGE LDS Sample buffer (pH > 7.0 at 70 degrees C)
-Reduced state of the proteins maintained during electrophoresis and blotting of the proteins by the NuPAGE Antioxidant
Please refer to the following paper: Moos M Jr, Nguyen NY, Liu TY (1988) Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support. J Biol Chem 263:6005-6008.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

If using Invitrogen Semi-Dry Blotter (Cat. No. SD1000), follow instructions in the manual for it.

Here is the transfer protocol optimized for the Bio-Rad Semi-Dry Transfer Unit. NuPAGE transfer buffer can be used for Tris-Glycine gels.

1) Working transfer buffer: 10% methanol, 1:1,000 Antioxidant in 2X NuPAGE transfer buffer (Bis-Tris 50 mM and Bicine 50 mM). If you need to prepare 100 mL of the working buffer from the NuPAGE 20X Transfer Buffer (Cat. No. NP0006), mix the following: 10 mL of 20X transfer buffer, 10 mL of MeOH, 100 µL of Antioxidant, 80 mL of DI H2O.

2) Filter papers: The transfer buffer-soaked filter papers of the sandwich are the only reservoir in the Semi-Dry Transfer Cell. If Invitrogen pre-cut membrane/filter sandwiches are used, at least 2 extra filter papers (0.4 mm/filter in thickness) on each side of the gel (or membrane) are required. When assembling one gel/membrane sandwich, presoak 6 Invitrogen filter papers (or 2 thicker filter papers) and 1 membrane in working transfer buffer (prepared in step 1) and sandwich them on the top of the anode plate as follows: filter paper--filter paper--filter paper--membrane--gel--filter paper--filter paper--filter paper

3) Blotting conditions: We found 15 V for 15-30 min is optimal for NuPAGE transfer buffer in the Bio-Rad Semi-Dry Transfer Cell. Semi-dry transfer units from other manufacturers should be used according to unit's instructions.

4) For transfer of large proteins (100 kD or larger), pre-equilibrate the gel with 0.02-0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich. Please note that transferring Tris-Glycine gels using NuPAGE transfer buffer in the Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell may be less efficient than using Tris-Glycine transfer buffer (Cat. No. LC3675) in the XCell II blot module (semi-wet).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Yes. The NuPAGE transfer buffer works with Tris-Glycine gels. Proteins are subjected to a more neutral pH, and the absence of glycine in the NuPAGE transfer buffer makes protein sequencing of proteins extracted from the gels much easier. While protein transfer is generally more efficient when using the NuPAGE transfer buffer, remember that the other benefits of the NuPAGE system (band sharpness, better resolution, sample stability) only apply if the proteins have been separated under these conditions prior to transfer (i.e., on a NuPAGE gel).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

This is perfectly acceptable with the XCell II Blot Module. The liquid in the outer buffer chamber only serves as a coolant or heat sink. The reason why water is recommended is because it is a less expensive alternative.

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If the buffer deviates from its expected pH by 0.2 units or higher, discard and remake the buffer after rechecking the reagents and the water purity. Do not adjust the pH with acid or base, as this will increase the conductivity of the buffer and result in higher current during the transfer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

We recommend the following transfer conditions: 30 V constant for 1 hr with the expected current of 220 mA/gel (start of run) to 180 mA/gel (end of run). For an overnight transfer, we recommend using 1-15 V constant. Please refer to the Nupage Large Protein Analysis System manual for more information (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/largeproteinanalysis_man.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

We recommend discarding the buffer and remaking it after rechecking the reagents and the water purity. We do not recommend adjusting the pH with acid or base, as this will increase the conductivity of the buffer and result in higher current during the transfer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Chlorobutanol is used as a preservative in the NuPAGE transfer buffer and is not necessary for efficient transfer of proteins. You may prepare the buffer without chlorobutanol but keep in mind that the buffer will not be stable for long periods. We recommend using it within 2 weeks.

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We recommend adding 10% methanol to the NuPAGE transfer buffer for transfer of one gel and 20% methanol for the transfer of 2 gels.

Find additional tips, troubleshooting help, and resources within our Protein Gel 1D Electrophoresis Support Center.

Yes, we recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer for enhanced blotting results with reduced proteins in order to maintain the reduced state of the proteins throughout the run.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Yes, the NuPAGE Transfer Buffer protects against modification of the amino acid side chains and is compatible with N-terminal protein sequencing using Edman degradation.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.