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For nondenaturing RNA electrophoresis, we recommend using our E-Gel Precast Agarose Gels. Please note that E-Gel Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.

For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.

For denaturing RNA electrophoresis, our E-Gel EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:

RNA Loading Buffer:
Deionized formamide: 200 µL
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10 mM EDTA): 40 µL
Deionized formaldehyde: 76 µL
Water: 14 µL

Denaturing Electrophoresis Conditions:
1. Mix 15 µL of RNA loading buffer with 1-5 µL of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel EX agarose gel.
5. Electrophorese for 30 minutes.

For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

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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.

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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.

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Here are possible causes and solutions:

- Improper labeling. Make sure that the labeling protocol is correctly followed to obtain the best results. Make sure you have added the Lumio Green Detection Reagent to the samples prior to electrophoresis. Limit exposure of the Lumio Gel Sample Buffer (4X) to air. Always return the Lumio Green Reagent and Lumio Enhancer to ?20 degrees C immediately after use to preserve the activity of buffers.
- Low protein load or low expression level. Check total protein loaded on the gel by staining the gel with a total protein stain as described in the manual. Load at least 1 pmole of the Lumio fusion protein. Make sure the Lumio tag is in frame and the protein is expressed properly. A positive control is supplied with the Lumio vectors to verify the expression protocol.
- The gel is exposed to UV light for a long time. The fluorescent dye of the Lumio Green Reagent is sensitive to photobleaching, so avoid exposing the gel to UV light for a long time.
- The gel is not visualized immediately or imaged properly. Be sure to visualize the gel after removing the gel from the cassette and view the gel immediately after electrophoresis. Use a UV transilluminator or a laser-based scanner using appropriate filters as described in the manual. Tip: If you have run BenchMark Fluorescent Protein Standard on the same gel and can view the standard bands on the gel, then you are imaging the gel properly.

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

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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.

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BCA Reducing Agent Compatible Protein Assay (Cat. No.s 23250, 23252), 1-D gel electrophoresis, 2-D gel electrophoresis (dilute sample 1:10 in sample buffer), Western blot, immunoprecipitation, ion exchange chromatography, polyhistidine-tag protein purification, and activity assays.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

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1) Trim excess plastic from IPG strip

2) Equilibrate IPG strip for 2 x 15 min in 5 mL of the appropriate buffer (Use NuPAGE sample buffer for NuPAGE ZOOM gels and Tris Glycine ZOOM gels). Equilibration buffer: 1X NuPAGE sample buffer, 50 mM DTT.

3) Place equilibrated IPG strip in large well of ZOOM gel. Seal the strip in place with an agarose (0.5%) overlay.

4) Load molecular weight markers in small well of ZOOM gel.

Variations: To alkylate the proteins after reducing them, prior to separation on ZOOM gels: 1) Incubate IPG strip for 15 min in equilibration buffer 2) Transfer strip to Equilibration Buffer containing 125 mM iodoacetamide and lacking reducing agent. Incubate an additional 15 min. To denature the proteins with urea after IEF: 6 M urea can be added to Equilibration Buffer, if desired.

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

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Possible cause:

*Excess reducing agent (beta-mercaptoethanol)
*Skin protein contaminants (keratin)

Remedy:

*The addition of iodoacetamide to the equilibration buffer just before applying the sample to the gel has been shown to eliminate these artifact bands.
*Use new electrophoretic solutions and wear gloves when handling and loading the gel. This issue is more common when highly sensitive stains are used.

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

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There is no fixation step in the Thermo Scientific 6xHis Protein Tag Stain Reagent Set staining procedure. Hence, staining with this kit does not inhibit subsequent total protein staining with general protein stains, or electrophoretic transfer to membrane. Note: Bis-Tris gels run in MOPS or MES buffer may require fixing in 50% methanol: 7% acetic acid for 15 minutes before performing the stain procedure. After electrophoresis, fix the gel and then proceed with Step 1 of the procedure.

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

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A forward smear indicates that the antibody was being reduced in the gel during migration. This could have been caused by the addition of antioxidant in the sample buffer or due to rearrangement of disulfide bonds during heating in NuPAGE Sample buffer. Make sure that the correct concentration of reducing agent is used in the sample buffer and do not add any antioxidant in the sample buffer.

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

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Here are possible causes and solutions:

- The buffer was accidentally made too dilute, therefore increasing resistance and thus lowering conductivity and current: Check the transfer buffer and its reagent components and then re-dilute it or remake it.
- The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply: Check the equipment.
- There is a leak in the blot module (this is indicated by a drastic decrease in current and in buffer volume within the module): Ensure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer.
- Tape at the bottom of the gel cassette was not removed: Double check that the tape on the bottom of the gel has been removed.

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

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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.

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

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Incubating ZOOM Strips in NuPAGE LDS Sample Buffer equilibrates the strips in SDS buffer and prepares the strips for 2D SDS-PAGE.

We recommend using the NuPAGE LDS Sample Buffer containing 50 mM DTT (NuPAGE Sample Reducing Agent) with NuPAGE Invitrogen 4-12% Bis-Tris ZOOM Gels and Invitrogen 4-20% Tris-Glycine ZOOM Gels. You need 5-15 mL of buffer per equilibration tray. For alkylation, we recommend incubating the strips in 125 mM Alkylating Solution (prepared by dissolving 232 mg of fresh iodoacetamide in 10 mL of 1X NuPAGE LDS Sample Buffer).

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

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Yes, formamide is supplied as a liquid even though it is sold by weight.

It is appropriate to use out of the bottle for most applications (hybridization buffer, loading buffers for sequencing gels, and formaldehyde-containing RNA gels) if less than one year old and stored properly (-20 degrees C). (If formamide smells like ammonia, it is probably breaking down and should not be used.) However, we have seen that in a protocol we have for running RNA gels without formaldehyde in the gel, freshly deionized formamide is essential for preparation of the loading buffer. That protocol as well as the protocol for deionizing the formamide follows:

These reagents are used in electrophoresis of RNA in agarose gels in 1X MOPS EDTA buffer. (Note: no formaldehyde is used in the gel.) The sample is prepared in the sample buffer (1.5 to 3 µL sample mixed with sample buffer). Heat the RNA in sample buffer for 10 min at 65 degrees C. Place on ice immediately then load gel.

10X MOPS EDTA (10X ME)

(1) Measure out 700 mL DEPC-treated water in a DEPC-treated 1-liter graduated cylinder and place in a DEPC-treated 1-liter beaker.
(2) Place beaker on stir plate and begin stirring with a DEPC-treated stir bar.
(3) Add 104.65 g MOPS to beaker. Weigh out MOPS using a sterile-flamed spatula from an unopened bottle MOPS or one set aside for RNA only. Dissolve by stirring.
(4) Add 40 mL 0.25 M EDTA to solution. Note: the EDTA solution should be made with DEPC-treated processed water.
(5) Adjust pH to 7.0 by adding 10 N NaOH (~20 mL)
(6) Bring up to 1 liter volume with DEPC-treated water.
(7) Filter solution using a 0.2 µm Nalgene filter unit.
(8) Store at 4 degrees C for six months in a DEPC-treated glass bottle. (This prevents solution from yellowing.) NOTE: The presence of yellow color does not affect the performance of the buffer.

Formamide Deionization protocol:
1. Add 1 g mixed bed, ion exchange resin for every 10 ml of formamide. Suitable ion exchange resins include BioRad AG501-X8 and Fisher Resin 1-300.
2. Stir for 30 to 60 min @ RT.
3. Filter through Whatman No. 1 filter paper.
4. Dispense into units of use and store at -20°C

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

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

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