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NuPAGE™ Antioxidant - FAQs

NuPAGE™ Antioxidant - FAQs

View additional product information for NuPAGE™ Antioxidant - FAQs (NP0005)

18 product FAQs found

Can I prepare my protein sample with the reducing agent and store it for future use?

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.

My LDS or SDS sample buffer precipitates when stored at 4 degrees C. Can I warm it up? Can I store it at room temperature?

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.

How are Bolt gels different than NuPAGE gels?

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.

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

What is the advantage of NuPAGE Gels over regular Tris-Glycine gels?

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.

What does the NuPAGE Antioxidant do? How much do I add to the NuPAGE running buffer?

The reducing agents DTT and beta-mercaptoethanol will not migrate through the gel with the sample in the neutral pH environment of the NuPAGE Gels. Instead, the reducing agent tends to remain at the top of the gel. Disulfide bonds are less reactive at neutral pH and are less likely to reoxidize than in a higher-pH system. However, in the absence of an antioxidant some reoxidization may occur during the electrophoresis, resulting in slightly diffuse bands.

The NuPAGE Antioxidant (a proprietary reagent) is added to the running buffer in the upper (cathode) buffer chamber only when performing electrophoresis under reducing conditions. The NuPAGE Antioxidant migrates with the proteins during electrophoresis preventing the proteins from reoxidizing and maintaining the proteins in a reduced state. The NuPAGE Antioxidant also protects sensitive amino acids such as methionine and tryptophan from oxidizing.

We also recommend using the NuPAGE Antioxidant with reduced samples that have been alkylated, for optimal results.

The NuPAGE Antioxidant is NOT compatible with gel systems other than the NuPAGE system as the antioxidant is not efficient at higher pHs of other gel systems.

0.5 mL of antioxidant is added to 200 mL (400x dilution) of running buffer and placed in the upper buffer chamber.

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

Is it possible to run reduced and non-reduced samples on the same NuPAGE gel? Should the Antioxidant be used?

If the Antioxidant is omitted from the running buffer, it is possible to resolve reduced and non-reduced samples on the same gel, although the resolution may be lower. Furthermore, it is not recommended that the reduced and non-reduced samples be run side-by-side in adjacent lanes.
However, because of the neutral pH of the NuPAGE gels, the reducing agent (beta-mercaptoethanol or DTT) will not migrate through the gel with the protein the way it does in the basic environment of the Tris-Glycine gels. Instead, the reducing agent tends to remain at the top of the gel. For this reason, the NuPAGE Antioxidant is incorporated into the buffer in the upper buffer chamber. The antioxidant is able to migrate fully with the proteins and keep them reduced. As a result, it is possible that proteins prepared as non-reduced samples could become somewhat reduced during the electrophoresis run. This would result in smearing of the samples.

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

Is there a reasonable replacement for the NuPAGE antioxidant for gel electrophoresis?

One can use 0.002% thioglycolic acid in the upper buffer reservoir. This is a good scavenger of free radicals. The reference to this is described by Hunkapiller et al, Methods of Enzymology, (91), 399, 1983. Caution should be taken when using this method since this compound is both toxic and expensive. In addition, the TGA must be fresh as it tends to become oxidized itself over time. Oxidized TGA will actually promote sample re-oxidation.

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

Are the NuPAGE buffers/components (i.e., sample buffer, running buffer, and antioxidant) compatible with Bolt gels?

Yes, NuPAGE buffers/components are compatible with Bolt gels.

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

Do I need to add the chlorobutanol when making the 20X NuPAGE Transfer Buffer?

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.

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

Can I transfer NuPAGE gels using Carbonate or CAPS transfer buffers?

We do not recommend using Carbonate or CAPS transfer buffers to transfer NuPAGE gels as the transfer efficiency will be badly compromised. Further, the high pH environment (>pH 9) of these buffers will make the NuPAGE Antioxidant non-functional.

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

Do you have any tips to improve transfer of high molecular weight proteins from NuPAGE gels?

To increase efficiency of transfer of high molecular weight proteins from NuPAGE gels, we recommend pre-equilibrating the gel in 2x NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1x NuPAGE transfer buffer containing methanol and 0.01% SDS.

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

Do you recommend adding the NuPAGE Antioxidant to the NuPAGE transfer buffer when I transfer proteins from NuPAGE Bis-Tris or NuPAGE Tris-Acetate gels?

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.

Can I use the NuPAGE Antioxidant with gel systems other than NuPAGE gels, e.g., Tricine gels?

No. It is not efficient at the higher pH values of the other gel systems.

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

I am planning to run a NuPAGE gel with reduced samples. Why is it necessary to add NuPAGE Antioxidant to the running buffer in the upper buffer chamber?

We recommend adding the NuPAGE antioxidant to the running buffer in the upper buffer chamber to keep samples reduced/bands tight throughout the run. At the neutral pH of the NuPAGE gels, the reducing agent tends to stay at the top of the well and not fully migrate throughout the gel. The antioxidant compensates for this by migrating fully with the proteins and keeping them reduced throughout the run. We recommend adding 0.5 mL of antioxidant to 200 mL (400X dilution) of running buffer and placing it in the upper buffer chamber.

Note: The antioxidant, by itself, is not efficient enough to completely reduce proteins. For complete reduction, samples must be treated with reducing agent prior to loading.

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

I stored the NuPAGE antioxidant at 4 degrees C and it seems to have formed a precipitate. Can I still use it?

The NuPAGE antioxidant has a tendency to precipitate when stored in the cold (4 degrees C). It will go right back into solution if gently warmed.

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

Should I use the NuPAGE Antioxidant when I run reduced protein samples in Bolt Bis-Tris Plus gels?

It is not necessary to use the Antioxidant while running reduced samples in Bolt Bis-Tris Plus gels but wouldn't hurt to use it.

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