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  6. Invitrogen™ NuPAGE™ Sample Reducing Agent (10X)

Invitrogen™ NuPAGE™ Sample Reducing Agent (10X)

Catalog No. NP0009
Encompass
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NP0004 250μL
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Description

Includes

NuPAGE Sample Reducing Agent (500mM dithiothreitol)

Ready-to-use reagent to reduce protein samples for protein gel electrophoresis

NuPAGE Sample Reducing Agent contains 500mM dithiothreitol (DTT) at a ready-to-use, 10X concentration in a stabilized liquid form. Use NuPAGE Sample Reducing Agent when running proteins on NuPAGE Novex Bis-Tris gels to maintain protein samples in a reduced state during electrophoresis.

1D Gel Electrophoresis, 2D Gel Electrophoresis, IsoElectric Focusing, Protein Gel Electrophoresis, Protein Sample Fractionation, Protein Sample Preparation and Protein Purification, Proteins, Expression, Isolation and Analysis

Order Info

Shipping Condition: Wet Ice

Specifications

Content And Storage NuPAGE™ Sample Reducing Agent (500 mM dithiothreitol) should be stored at 4°C. Guaranteed stable for 6 months unless otherwise specified in the product literature.
Concentration 500 mM
Quantity 10mL
Shipping Condition Wet Ice
Product Line NuPAGE
Product Type Sample Reducing Agent

Frequently Asked Questions (FAQs)

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.
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.
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.
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.
Where do I find buffer recipes for your precast protein gels?

The formulations of buffers for our precast protein gels can be found at this link: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-electrophoresis-buffers-reagents.html
Can beta-mercaptoethanol (BME) be used rather than DTT as the reducing agent in the NuPAGE LDS Sample Buffer?

Either BME or DTT can be used in the NuPAGE LDS Sample Buffer.

Make sure that a fresh solution of BME is used. FINAL concentration:

DTT 50-100 mM

BME 2-5%
Can other reducing agents other than DTT or BME be used to reduce proteins prior to electrophoresis? For example, what about TCEP (Tris Carboxy Ethyl Phosphene)?

TCEP, Tris Carboxy Ethyl Phosphene is an alternative sulfhydryl reducing agent for protein samples. It is an extremely potent and effective reducing agent for particularly ‘difficult' proteins. It is compatible with the Tris-Glycine gels and NuPAGE gels. It should be added to the sample buffer for these systems. 20 mM final (maximum) concentration is sufficient for samples. You may add alkylating agents, e.g. Iodine (50 mM Iodoacetic acid), to prevent re-forming of S-S bonds but it is not necessary. Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.
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.
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.
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.
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.
Can I use beta-mercaptoethanol instead of the NuPAGE Sample Reducing agent?

Although we recommend using the NuPAGE Sample Reducing agent for stability reasons, fresh, neat beta-mercaptoethanol can be substituted for the NuPAGE Sample Reducing Agent, with equivalent results. A final concentration of 2-5% beta-mercaptoethanol is usually sufficient to reduce the sample.

What is the composition of the NuPAGE Sample Reducing agent?

The NuPAGE Sample Reducing agent (10X) contains 0.5M DTT (Dithiothreitol) with proprietary stabilizers .

How should I store the NuPAGE Sample Reducing agent and NuPAGE Antioxidant?

NuPAGE Reducing Agent and NuPAGE Antioxidant should be stored at 4 degrees C.

For Research Use Only. Not for use in diagnostic procedures.