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General 1D Electrophoresis

Bolt™ Bis-Tris Plus Gels

NuPAGE™ Bis-Tris and NuPAGE™ Tris-Acetate Gels

Novex™ Tris-Glycine Gels

Thermo Scientific™ Precise™ Protein Gels

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General 1D Electrophoresis

Our precast protein gels do not contain SDS but they can be run under denaturing conditions when used with the appropriate denaturing running buffer. Please see the table below to determine which buffer to use with each type of gel:

Gel type

Native electrophoresis 

Denaturing electrophoresis

 

Running buffer to use

Novex™ Tris-Glycine gels

 

Novex™ Tris-Glycine Native Running Buffer

Novex™ Tris-Glycine SDS Running Buffer

 

NuPAGE™ Tris-Acetate gels

 

Novex™ Tris-Glycine Native Running Buffer

NuPAGE™ Tris-Acetate SDS Running Buffer

 

NuPAGE™ Bis-Tris gels

 

_

NuPAGE™ MOPS-SDS Running Buffer or NuPAGE™ MES-SDS Running Buffer

 

Bolt™ Bis-Tris Plus gels

 

_

Bolt™ MOPS SDS Running Buffer or Bolt™ MES SDS Running Buffer

 

Thermo Scientific™ Precise™ Tris-Glycine gels 

 

Tris-Glycine SDS Running Buffer without SDS added

Tris-Glycine SDS Running Buffer

Thermo Scientific™ Precise™ Tris-HEPES gels 

 

_

Tris-HEPES SDS Running Buffer

 

Note: NuPAGE™ Bis-Tris gels, Bolt™ Bis-Tris Plus gels, and Thermo Scientific™ Precise™ Tris-HEPES gels cannot be run under native conditions; they can only be run under denaturing conditions.

The recommended sample loading volumes and protein load per band for the different well formats/gels can be found as shown below:

Our Novex™ precast protein gels contain a stacking gel that is ~8 to 9 mm long (it ends right above the first ridge on the cassette). The manufacturing method used results in an interface between the stacking and resolving gels that is not visually detectable.

The percentage of the stacking gel is 4% in most of our gels including the Bolt™ Bis-Tris Plus gels. The NuPAGE™ Tris-Acetate gels contain a 3.2% stacking gel.

Here is the information:

Gel Acrylamide:bisacrylamide % Crosslinker
Tris-Glycine gels (except 4% Tris-Glycine gels) 37.5:1 2.6%
4% Tris-Glycine gels 76:1 1.3%

 

We do not recommend storing reduced protein samples for long periods of time even if they are frozen because reoxidation of the sample may happen during storage, causing inconsistent results.

We do not recommend running reduced and non-reduced protein samples on the same gel, especially in adjacent lanes, since the reducing agent may have a carry-over effect on the non-reduced samples if they are in close proximity.

We recommend using the NativeMark™ Unstained Protein Standard, Cat. No. LC0725.

No, two gels will run at the same constant voltage but the current will double.

All our Novex™ protein gels are available in Mini format. Certain gel chemistries (NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, and Novex™ Tris-Glycine gels) are also available in the wide Midi format. Note that Bolt™ Bis-Tris gels are not available in the Midi format. Our Thermo Scientific™ Precise™ precast gels are only available in Mini format.

All our Novex™ precast protein gels (Novex™ gels, NuPAGE™ gels, and Bolt™ Bis-Tris Plus gels) are available in Mini format (Cassette: 10 cm x 10 cm; Gel: 8 cm x 8 cm). Please note that our Original Bolt™ Bis-Tris Plus Mini gels (discontinued as of December 31, 2014) had slightly different dimensions (Cassette: 10 cm x 10.5 cm; Gel: 8 cm x 8.3 cm).

Our NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, and Novex™ Tris-Glycine gels are also available in the wider Midi format. Note that Bolt™ Bis-Tris Plus gels are not available in the Midi format.

Our Thermo Scientific™ Precise™ precast gels are only available in Mini format.

Please see the table below for gel dimensions of Mini and Midi gels of different chemistries:

  Midi gels Mini gels
  NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, & Novex™ Tris-Glycine NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, & Novex™ Tris-Glycine Bolt™ Bis-Tris Plus (Cat. No. NWxxxxBOX) *Original Bolt™ Bis-Tris Plus (Cat. No. BGxxxxBOX) Thermo Scientific™ Precise™ Tris-Glycine Thermo Scientific™ Precise™ Tris-HEPES
Gel dimension 13 cm x 8.3 cm 8 cm x 8 cm 8 cm x 8.3 cm 8 cm x 8.3 cm 6.8 cm x 8 cm 8.8 cm x 8 cm 5.8 cm x 8 cm
Cassette dimension 15 cm x 10.3 cm 10 cm x 10 cm 10 cm x 10 cm 10 cm x 10.5 cm 8 cm x 10 cm 10 cm x 10 cm 8.5 cm X 10 cm

Our Original Bolt™ Bis-Tris Plus Mini gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) can only be run in the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014, and will be offered until inventory is depleted).

Our New Bolt™ Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX), as well as our Novex™ Mini gels and NuPAGE™ Mini gels can be run using the Mini Gel Tank, or XCell SureLock™ Mini-Cell. To run these gels using the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014), upgrading of the tank is necessary by replacing the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video.

Our Midi gels can be run using the XCell4 SureLock™ Midi-Cell.

Bolt™ Bis-Tris Plus gels are not available in the Midi format.

Midi gels are wider than Mini gels and hence have a larger number of wells to accommodate additional samples in one gel. An experiment from a Mini gel can be easily scaled-up to a Midi gel of the same gel chemistry. Please see the table below for gel dimensions of Novex™ Mini and Midi gels of different chemistries:

Midi gels Mini gels
NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, & Novex™ Tris-Glycine NuPAGE™ Bis-Tris, NuPAGE™ Tris-Acetate, & Novex™ Tris-Glycine New Bolt™ Bis-Tris Plus (Cat. No. NWxxxxxBOX) *Original Bolt™ Bis-Tris Plus (Cat. No. BGxxxxxBOX)
Gel dimension Cassette dimension Gel dimension Cassette dimension Gel dimension Cassette dimension Gel dimension Cassette dimension

13 cm x 8.3 cm

15 cm x 10.3 cm

8 cm x 8 cm

10 cm x 10 cm
  8 cm x 8.3 cm
10 cm x 10 cm

8 cm x 8.3 cm

10 cm x 10.5 cm

We do not recommend recycling our plastic cassettes because they have a chemical coating on them that may produce toxic fumes when melted and potentially cause contamination.

The cassettes are made of a styrene copolymer.

All Novex™ protein gels contain sucrose as a density-adjusting agent to facilitate pouring of the gel. Protein samples run on Novex™ gels would be contaminated with large amounts of sucrose. Thus, Novex™ gels are not recommended for this application.

All detergents, or even phospholipids in cell extracts, will form mixed micelles with SDS and migrate down into the gel. They can also interfere with the SDS:protein binding equilibrium. Most of the non-ionic detergents, including NP-40, are the worst at interfering with SDS-PAGE. The rule of thumb is to keep the ratio of SDS to lipid or other detergent at 10:1 or greater to minimize these effects.

Midi gels can be transferred using:

  • iBlot™ Dry Blotting System in conjunction with Transfer Stacks
  • Novex™ Semi-Dry Blotter for simultaneous transfer of up to 2 Midi-gels
  • Thermo Scientific™ Pierce™ Power Blotter for simultaneous transfer of up to 2 Midi gels
  • Thermo Scientific™ Pierce™ G2 Fast Blotter (will be discontinued as soon as we exhaust current inventory).

To run Mini gels with 10 cm gel cassettes using a Bolt™ Mini Gel Tank (without replacement of 10.5 cm cassette clamp cam handles with 10 cm cassette clamp cam handles), please use the instructions provided on Page 22 of the manual.


Note: For optimal results, to run 10 cm cassette Mini gels with a Bolt™ Mini Gel Tank, one should replace the black 10.5 cm cassette clamp cam handles on the Bolt™ Mini Gel Tank with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video. Additional resources can be found here.

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. 20mM final (maximum) concentration is sufficient for samples. You may add alkylating agents (e.g. Iodine, to prevent re-forming of S-S bonds) but it is not necessary (50mM Iodoacetic Acid). Do not heat because this will hydrolyze much of your sample. Instead let the sample sit for several minutes at RT and then load.

No, CTAB will not work with any of our gels except for the NuPAGE™ Tris-Acetate gels. To use CTAB, you would need to use a running buffer of 50mM acetic acid and 50mM beta-alanine in equal concentrations. You would also need to switch the electrodes. Since CTAB is a cationic detergent, this would establish conditions for running a basic protein towards the anode (into the gel).

There shouldn't be any negative effects unless the percentage of acetonitrile reaches 40 or 50% of the sample size. At this point, there is the possibility of the acetonitrile affecting the binding of SDS to the protein, which in turns affects the migration of the protein.

Phenol red was originally included in the Novex™ sample buffers as a true ion-front tracking dye for the very small pore-sized gels, especially Tricine gels. Phenol red is smaller than Serva Blue G250. In high percentage gels, molecules in this size range are resolved on the basis of size, such that phenol red runs with the ion front while G250 can run as much as 2 cm behind the ion front. Conversely, in larger-pored gels, there is no size-based resolution in the size range of these dyes, and the dyes resolve on the basis of charge density, with G250 running at the ion front and phenol red migrating more slowly. Phenol red is not particularly useful in the NuPAGE™ system, even though it is included in the NuPAGE™ sample buffer for the sake of consistency, especially with the Novex™ standards, which all contain phenol red.

Using constant voltage allows the current and power to decrease during the run, providing a safety margin in case of a break in the system. Having lower power is a safety benefit and will also decrease the chances of experiencing an overheating of the gel. Further, the constant voltage setting does not need adjustment to account for differences in number or thickness of gels being run.

If you are running the gels at constant voltage, you do not need to increase the voltage regardless of the number of gels. However, the resulting current and wattage observed will multiply linearly with the number of gels. Keep in mind that the expected total current for your gels should not exceed the current limit of the power supply, or else the current will plateau and the run will slow down. (For example: Recommended constant voltage for running a NuPAGE™ Bis-Tris gel with MES Buffer is 200 V, with a starting current of 110-125 mA/gel and end current of 70-80 mA/gel. If the power supply has a current limit of 500 mA, the maximum number of NuPAGE™ Bis-Tris gels that can be run at one time with full power is 500 mA/125 mA = 4 gels. Any additional gels will decrease the current per gel and increase the run time.

If you are running the gel at constant voltage, you do not need to increase the voltage regardless of the thickness of the gel.

This is not really recommended, but it is always possible to increase run time by lowering the voltage of the run. In general, the relationships are linear – i.e. decreasing voltage by half will double the run time.

We do not recommend using gels past their expiration date because over time, the polyacrylamide hydrolyzes to acrylic acid and ammonia and this will affect the resolution of the proteins. Breakdown of polyacrylamide matrix is identified by:

- Ghost bands and doublets, seen first in the high molecular weight proteins

- Smiling of dye front across the gel, with bands in outer lanes becoming very slanted - proteins run slower there due to change in pH and pore size over time.

Having everything at room temperature means not having to thaw reagents, thus saving time in gel casting.

Yes, SureCast Glass Plates can be reused. They are up to 22 times more durable than Bio-Rad glass plates.

The SureCast Handcast System and reagents are available both as a package or as standalones.

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

The Laemmli buffer or 2X SDS Buffer is composed of the following: 100 mM Tris HCl , pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol. 2X SDS gel loading buffer lacking dithiothreitol can be stored at room temperature. Dithiothreitol should then be added, just before use.

Bolt™ Bis-Tris Plus Gels

While they are both Bis-Tris based gels, the chemistries are quite different. Bolt™ Bis-Tris Plus gels are designed for optimized western blot performance. They deliver well-resolved, sharp, straight bands with higher band volume enabled by their wedge well design.

Bolt™ Bis-Tris Plus gels have a proprietary acrylamide/bisacrylamide formulation that is based on the NuPAGE™ Bis-Tris gel chemistry but different from it.

While they are both Bis-Tris based gels, the chemistries are quite different. Bolt™ Bis-Tris Plus gels are designed for optimized western blot performance. They deliver well-resolved, sharp, straight bands with higher band volume enabled by their wedge well design.

We recommend storing them at 4–25 degrees C. They should not be frozen.

Bolt™ Bis-Tris Plus gels have a shelf life of 16 months when stored at 4–25°C.

Bolt™ Bis-Tris Plus gels contain a 4% stacking gel.

Our Original Bolt™ Bis-Tris Plus gels (Cat. No. BGxxxxxBOX), discontinued as of December 31, 2014) do not fit in the XCell SureLock™ Mini-Cell. They need to be run in the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014). Our New Bolt™ Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX) can be run using the XCell SureLock™ Mini-Cell (or Mini Gel Tank, Cat. No. A25977). To run these gels using the Bolt™ Mini Gel Tank, upgrading of the tank is necessary by replacing the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video.

Please note that the Bolt™ Mini Gel Tank has been discontinued as of December 31, 2014 and will be available for purchase until inventory is depleted. The Mini Gel Tank is an improved version of the Bolt™ Mini Gel Tank with enhancements in design allowing for:

  • Versatility—compatible with Bolt™ Bis-Tris Plus gels, NuPAGE™ Mini gels, and Novex™ Mini gels
  • Easy sample loading—with a forward-facing configuration
  • Less running buffer required— with two separate gel chambers, just add buffer for each gel up to the marked fill line
  • Simultaneous visualization of both gels—streamlined, side-by-side tank configuration
  • Simplified monitoring of prestained markers—with new white tank stand

Please check out this video for operating the Mini Gel Tank.

Bolt™ Bis-Tris Plus gels have a 1 mm thickness.

Our New Bolt™ Bis-Tris Plus gels (Cat. No. NWxxxxxBOX) have the following dimensions:

  • Cassette: 10 cm x 10 cm
  • Gel: 8 cm x 8 cm

Our Original Bolt™ Bis-Tris Plus gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) had slightly different dimensions:

  • Cassette: 10 cm x 10.5 cm
  • Gel: 8 cm x 8.3 cm

The main difference is in the dimensions of the gels. Our New Bolt™ Bis-Tris Plus gels (Cat. No. NWxxxxxBOX) have the following dimensions:

  • Cassette: 10 cm x 10 cm
  • Gel: 8 cm x 8 cm

The new Bolt™ Bis-Tris Plus gels can be run using the new Mini Gel Tank (Cat. No. A25977), or the XCell SureLock™ Mini-Cell. To run these gels using the Bolt™ Mini Gel Tank (discontinued as of December 31, 2014), upgrading of the tank is necessary by replacing the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video.

Our Original Bolt™ Bis-Tris Plus gels (Cat. No. BGxxxxxBOX, discontinued as of December 31, 2014) had slightly different dimensions:

  • Cassette: 10 cm x 10.5 cm
  • Gel: 8 cm x 8.3 cm

Our Original Bolt™ Bis-Tris Plus Mini gels can only be run in the Bolt™ Mini gel tank (discontinued as of December 31, 2014, and will be offered until inventory is depleted).

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.

No. We only offer one size of 10 gels per box.

Bolt™ Bis-Tris Plus gels are not available in the Midi format.

SeeBlue™ prestained standard, SeeBlue™ Plus 2 prestained standard and Novex™ Sharp prestained standard are compatible with Bolt™ Bis-Tris Plus gels. Their migration is the same as that in NuPAGE™ gels with the corresponding buffer.

To upgrade the Bolt™ Mini Gel Tank to run the new Bolt™ Bis-Tris Plus gels, you have to replace the black 10.5 cm cassette clamp cam handles with gray 10 cm cassette clamp cam handles (Cat. No. A26732). Instructions for replacement of the cam handles can be found on Page 20 of the manual or in this video. Additional resources can be found here.

The composition of the 1X NuPAGE™ LDS Sample Buffer is as follows:

141 mM Tris base

106 mM Tris HCl

2% LDS

10% Glycerol

0.51 mM EDTA

0.22 mM SERVA Blue G

0.175 mM Phenol Red

pH 8.5

To prepare 10 mL of 4 X NuPAGE™ LDS Sample Buffer, dissolve the following reagents to 8 mL ultrapure water:

Tris HCl 0.666 g

Tris Base 0.682 g

LDS 0.800 g

EDTA 0.006 g

Glycerol 4 g

SERVA Blue G (1 solution) 0.75 mL

Phenol Red (1 solution) 0.25 mL

Mix well and adjust the volume to 10 mL with ultrapure water.

Store at 4°C. The buffer is stable for 6 months when stored at 4°C.

NuPAGE™ Bis-Tris and NuPAGE™ Tris-Acetate Gels

While the NuPAGE™ Bis-Tris gels do not contain SDS, they are only intended for denaturing gel electrophoresis conditions as both the NuPAGE™ MOPS SDS and NuPAGE™ MES SDS Running buffers contain SDS.

While the NuPAGE™ Tris-Acetate gels do not contain SDS, they can be used for denaturing gel electrophoresis when used in conjunction with NuPAGE™ Tris-Acetate SDS Running buffer and NuPAGE™ LDS Sample buffer. They can be used for native gel electrophoresis when used in conjunction with Novex™ Tris-Glycine Native Running buffer and Novex™ Tris-Glycine native sample buffer.

The composition of the NuPAGE™ Antioxidant is proprietary.

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.

We recommend storing them at 4–25 degrees C. They should not be frozen.

We recommend storing them at 4–25 degrees C. They should not be frozen.

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

NuPAGE™ buffers can be stored at 4–25 degrees C.

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

The pH of the NuPAGE™ 4X Sample buffer is much higher than that of the usual Laemmli Sample buffer: 8.5 vs. 6.8. At this pH (8.5), SDS will precipitate when added at the concentration needed for a 4X buffer. On the other hand, LDS (lithium dodecyl-sulfate) is more soluble and is just as effective for denaturation.

It would be okay to boil the protein sample as none of the buffer components are damaged upon boiling. The reason we recommend to heat the sample at 70 degrees C for 10 minutes and not boil the sample is because protein hydrolysis is reduced at lower temperatures.

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.

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.

NuPAGE™ gels have the following advantages over Tris-Glycine gels:

  • Higher stability and longer shelf life:
    • NuPAGE™ Bis-Tris gels and NuPAGE™ Tris-Acetate gels have a lower operating pH (pH 7 for NuPAGE™ Bis-Tris gels and pH 8.1 for NuPAGE™ Tris-Acetate gels) than Novex™ Tris-Glycine gels (pH 9.5). At basic pH, polyacrylamide hydrolyzes to polyacrylic acid and ammonia whereas at neutral pH, this hydrolysis is slower. Hence, NuPAGE™ gels have higher stability and longer shelf life than Novex™ Tris-Glycine gels (12 months at 4–25 degrees C for NuPAGE™ Bis-Tris gels and 8 months at 4 degrees C for NuPAGE™ Tris-Acetate gels vs 4–8 weeks at 4 degrees C for Tris-Glycine gels).
  • Better resolution of proteins due to:
    • Reduced undesired chemical modifications: Free acrylamide alkylates proteins at basic pH (8.5 to 9.0). It targets sulfhydryl cysteines and amine groups at the N-terminus and on lysines. This modification does not happen at pH below 8. Hence, proteins run on NuPAGE™ gels undergo fewer of these undesired chemical modifications than those run on Tris-Glycine gels.
    • Reduced hydrolysis of proteins: Heating of Tris-Glycine sample buffer (pH 6.8) results in a drop in pH, causing Asp-Pro cleavage of proteins. High temperature and longer duration of heating/boiling increase the rate of this cleavage resulting in multiple peptide bands of decreased intensity. At 100 degrees C, the pH drops as low as pH 4.3. On the other hand, NuPAGE™ LDS sample buffer (pH 8.5) drops to pH 8.1 when heated to 70 degrees C, avoiding this cleavage.
  • Faster run times (35–50 min for NuPAGE™ Bis-Tris gels and 1 hour for NuPAGE™ Tris-Acetate gels vs 90 min for Tris-Glycine gels)

The NuPAGE™ MOPS and MES SDS running buffers are compatible with Edman protein sequencing. Proteins can be sequenced directly from NuPAGE™ gels.

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

While they are both Bis-Tris based gels, the chemistries are very different as Bolt™ Bis-Tris Plus gels have been optimized for western blotting. Another key difference is the enabling wedge well design for Bolt™ Bis-Tris Plus gels, which allows larger sample volume load.

We do not recommend using NuPAGE™ Bis-Tris gels with NuPAGE ™MOPS or MES Running Buffer prepared without SDS for electrophoresis under native conditions. This buffer system may generate excessive heat, resulting in poor band resolution. Further, the protein of interest may not migrate very well in a neutral pH environment if it is not charged.

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.

NuPAGE™ gels are compatible with any of the standard Coomassie staining procedures. The protocols that are accelerated by heat are preferable as the heat serves as a “fix” for proteins, especially smaller peptides. NuPAGE™ gels are also compatible with most silver staining protocols. They are also compatible with copper or zinc staining, and fluorescent stains.

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.

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

NativePAGE™ Sample Buffers and Running buffers were developed specifically for use with the NativePAGE™ Bis-Tris gels. We do not recommend using these buffers for native applications with any other gels, including NuPAGE™ Tris-Acetate gels. For those gels, we recommend using the Novex™ Tris-Glycine Native Sample and running buffers.

We do not recommend using NativePAGE™ Sample and Running buffers with NuPAGE™ Bis-Tris gels. NuPAGE™ Novex Bis-Tris gels are optimized for denaturing conditions and have an extremely low operating pH, which makes it difficult for most proteins to migrate through them in a native state.

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.

Copper or zinc staining is a rapid, sensitive method for detection of protein bands . ~10ng reduced BSA on NuPAGE™ Bis-Tris gels can be detected with both the copper and zinc stain.

Copper Stain: Staining solution - 0.3M CuCl2

After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 ml of 1X running buffer* for 15 minutes. Immerse the gel in 100 ml of 0.3M CuCl2 solution for about 5 minutes (the protein band will appear as a negative stain with a blue background).

Zinc stain: Staining solution - 0.2 M Imidazole and 10 mM ZnCl2

After electrophoresis, remove the gel from the cassette and equilibrate the gel in 100 ml of 1X running buffer* for 15 minutes. Place the gel in 100 ml of 0.2M Imidazole solution for 10 minutes. Next immerse the gel in 100 ml of 10 mM ZnCl2 solution for about 5 minutes (the protein will appear as a transparent band with a white background).

*The 1X running buffer can be the buffer from the electrophoresis tank after run (MES, MOPS). However, for better contrast of the band, the 1X Tris-Glycine SDS running buffer is recommended.

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.

The temperature coefficient (Delta pKa/degree C) for Tris is -0.031/degree C. Therefore, if the pH of the NuPAGE™
LDS Sample Buffer is 8.5 at 25 degrees C, then its pH at 70 degrees C is 8.5 + (-0.031 X 45) = 7.1. Similarly, our Tris Glycine Sample Buffer at 85 degrees C would have a pH of 4.94, at 100 degrees C its pH would be 4.5.

Novex™ Tris-Glycine Gels

Novex™ Tris-Glycine polyacrylamide gel chemistry is based on the Laemmli system with minor modifications for maximum performance in the pre-cast format. These gels do not contain SDS and can therefore be used to accurately separate both native and denatured proteins by using the appropriate running buffer. Novex™ Tris-Glycine Gels provide reproducible separation of a wide range of proteins into well resolved bands.

The operating pH for Tris-Glycine gels is 9.5; the operating pH for NuPAGE™ Bis-Tris gels is 7 and for NuPAGE™ Tris-Acetate gels is 8.1.

NuPAGE™ gels have the following advantages over Tris-Glycine gels:

  • Higher stability and longer shelf life:
    • NuPAGE™ Bis-Tris gels and NuPAGE™ Tris-Acetate gels have a lower operating pH (pH 7 for NuPAGE™ Bis-Tris gels and pH 8.1 for NuPAGE™ Tris-Acetate gels) than Novex™ Tris-Glycine gels (pH 9.5). At basic pH, polyacrylamide hydrolyzes to polyacrylic acid and ammonia whereas at neutral pH, this hydrolysis is slower. Hence, NuPAGE™ gels have higher stability and longer shelf life than Novex™ Tris-Glycine gels (12 months at 4–25 degrees C for NuPAGE™ Bis-Tris gels and 8 months at 4 degrees C for NuPAGE™ Tris-Acetate gels vs 4–8 weeks at 4 degrees C for Tris-Glycine gels).
  • Better resolution of proteins due to:
    • Reduced undesired chemical modifications: Free acrylamide alkylates proteins at basic pH (8.5 to 9.0). It targets sulfhydryl cysteines and amine groups at the N-terminus and on lysines. This modification does not happen at pH below 8. Hence, proteins run on NuPAGE™ gels undergo fewer of these undesired chemical modifications than those run on Tris-Glycine gels.
    • Reduced hydrolysis of proteins: Heating of Tris-Glycine sample buffer (pH 6.8) results in a drop in pH, causing Asp-Pro cleavage of proteins. High temperature and longer duration of heating/boiling increase the rate of this cleavage resulting in multiple peptide bands of decreased intensity. At 100 degrees C, the pH drops as low as pH 4.3. On the other hand, NuPAGE™ LDS sample buffer (pH 8.5) drops to pH 8.1 when heated to 70 degrees C, avoiding this cleavage.
  • Faster run times (35–50 min for NuPAGE™ Bis-Tris gels and 1 hour for NuPAGE™ Tris-Acetate gels vs 90 min for Tris-Glycine gels

Our Tris-Glycine gels do not contain SDS. They can be run under native conditions by using the Novex™ Tris-Glycine Native Sample buffer and the Novex™ Tris-Glycine Native Running buffer. They can be run under denaturing conditions by using the Novex™ Tris-Glycine SDS Sample buffer and the Novex™ Tris-Glycine SDS Running buffer.

NativePAGE™ Sample Buffers and Running buffers were developed specifically for use with the NativePAGE™ Bis-Tris gels. We do not recommend using these buffers for native applications with any other gels, including Novex™ Tris-Glycine gels. For those gels, we recommend using the Novex™ Tris-Glycine Native Sample and running buffers.

Tris-Glycine gels are compatible with any of the standard Coomassie™ staining procedures. They are also compatible with most silver staining protocols.

Novex™ Tricine Gels are ideal for peptides and low molecular weight proteins (less than 10 kDa). Unlike Tris-Glycine gels, Tricine gels allow resolution of proteins with molecular weights as low as 2 kDa. Tricine, unlike glycine, will not interfere with sequencing, so Tricine gels are an excellent choice for direct sequencing after transferring to PVDF. In addition to good transfer efficiency, the Tricine system has a lower pH which minimizes unwanted protein modification. Tricine gels can only be run under denaturing conditions

Tris-Glycine gels may be transferred in CAPS buffer (10mM CAPS (3-cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0.

Yes, the new Novex WedgeWell gels can be run in the XCell SureLock Mini-Cell.

We use what we call ‘mirror printing’, the same way we do for Bolt™ gels. This means that when the gel is loaded in the Mini Gel Tank, the text will face in the readable direction (left to right).

We were able to optimize shelf life through a proprietary gel formulation change. Unfortunately, we are unable to provide specific details on the chemical changes.

The Novex WedgeWell gels can use standard Tris-Glycine sample and running buffers. For running under native conditions, we recommend using sample and running buffers that do not contain SDS (such as our Native Tris-Glycine premade buffers).

No. Novex WedgeWell gels must be stored at 4 degrees C.

Thermo Scientific™ Precise™ Protein Gels

Precise gels contain a 4% stacking gel.

The Precise Tris-Glycine gels are available in two different mini gel formats. The gel and cassette dimensions for the two formats are shown below:

  • Gel: 6.8 cm x 8 cm x 1 mm
    Cassette: 8 cm x 10 cm x 5 mm
  • Gel: 8.8 cm x 8 cm x 1 mm
    Cassette: 10 cm x 10 cm x 7 mm

Precise Tris-Glycine gels are stable for a year when stored at 4 degrees C.

Yes, the 10 cm x 10 cm x 7 mm cassette Precise Tris-Glycine gels are compatible with the XCell SureLock Mini Cell and the Mini Gel Tank.

The gel and cassette dimensions for the Precise Tris-HEPES gels are shown below:

  • Gel: 5.8 cm x 8 cm x 1 mm
  • Cassette: 8.5 cm x 10 cm x 4.5 mm

Precise Tris-HEPES gels are stable for a year when stored at 4 degrees C.

The pH of the Precise gels is pH 7.

No. Precise gels do not contain SDS. SDS is used to denature proteins and surround them with a uniform net negative charge. The charges on the protein become negligible and their shape no longer has an effect such that electrophoresis is conducted purely on size. Precise Gels need to be run with SDS. The SDS included in the sample and running buffers is enough to denature the proteins and maintain them in that state throughout the run.

Tris-HEPES Precise gels cannot be run under native conditions. They must be run with SDS in the Sample buffer and in the Tris-HEPES running buffer. The Tris-Glycine Precise gels can be run under both native and denaturing conditions.

The Precise Tris-HEPES gels are compatible with the XCell SureLock Mini Cell or Mini Gel Tank when used with adaptor plates.

Note: Two adaptor plates are required when running just one gel and one adaptor plate is required when running two gels using the XCell SureLock Mini Cell or Mini Gel Tank.

The Precise Tris-HEPES gels are designed to be used with the Tris-HEPES SDS running buffer for optimal speed and resolution. We do not recommend using them with Tris-Glycine SDS running buffer as this will result in poor migration and band resolution.

Yes. Precise gels can be air-dried. Dry your gels with a commercial drying solution if cracking is a problem. The recommended drying solution is 4% ethylene glycol in 35% ethanol.

All standard staining solutions work on Precise gels. However, best results are obtained with methanol concentrations of less than 30%. Coomassie stained gels can be destained with 6% acetic acid.

No. Precise gels can only be used with their appropriate buffers; Tris-HEPES-SDS Running Buffer for Tris-HEPES gels or Tris-Glycine-SDS Running Buffer for Tris-Glycine gels. This buffer system allows gels to run in 45 minutes. Using other buffers will have negative effects on performance.

Yes. The gel storage buffer does contain sodium azide which is washed away.

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