Invitrogen™ NuPAGE™ Transfer Buffer (20X)

Catalog No.	Quantity
NP0006	125mL
NP00061	1L

Catalog No. NP0006 Supplier Invitrogen™ Supplier No. NP0006

Description

Use to transfer proteins from NuPAGE Novex gels to membranes for Western blotting

NuPAGE Transfer Buffer can be used with the Novex Semi-Dry Blotter for semi-dry transfer or with the XCell II™ Blot Module for wet (tank) transfer. NuPAGE Antioxidant may be used with NuPAGE Transfer Buffer to enhance transfer of reduced proteins to membranes.

1D Gel Electrophoresis, Protein Gel Electrophoresis, Proteins, Expression, Isolation and Analysis, Western Blotting

Order Info

Shipping Condition: Room Temperature

Specifications

• Content And Storage: Store at room temperature. Guaranteed stable for 6 months unless otherwise specified in the product literature.
• Buffer: Transfer Buffers
• Concentration: 20X
• Gel Type: NuPAGE
• Gel Compatibility: NuPAGE™ Gels
• Quantity: 125mL
• Shipping Condition: Room Temperature
• Product Line: NuPAGE
• Product Type: Transfer Buffer

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.

How do I perform a semi-dry transfer of Tris-Glycine gels?

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).

Can I use the NuPAGE transfer buffer with Tris-Glycine Gels?

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).

When blotting with the XCell II Blot Module, what would happen if I filled the outer buffer chamber with transfer buffer instead of water?

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.

How much can the pH of the Western transfer buffer vary and still be effective?

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.

What conditions should I use to transfer my gel when using the NuPAGE Transfer buffer?

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).

The pH of my transfer buffer deviates from the recommended value by 0.2 units. Can I still use the buffer?

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.

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.

How much methanol do you recommend adding to the NuPAGE transfer buffer for transfer of NuPAGE Bis-Tris gels and NuPAGE Tris-Acetate gels?

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

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.

Is the NuPAGE Transfer buffer compatible with N-terminal sequencing of proteins via Edman degradation?

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.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

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