NuPAGE™ 3 to 8%, Tris-Acetate, 1.0 mm, Mini Protein Gel, 12-well, 2 Gels/Box - FAQs

View additional product information for NuPAGE™ Tris-Acetate Mini Protein Gels, 3 to 8%, 1.0–1.5 mm - FAQs (EA03755BOX, EA03752BOX, EA0378BOX, EA0375PK2, EA03785BOX, EA03752PK2, EA0375BOX)

71 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 it mean when bands appear to be getting narrower (or "funneling") as they progress down a protein gel?

There may be too much beta-mercaptoethanol (BME), sample buffer salts, or dithiothreitol (DTT) in your samples. If the proteins are over-reduced, they can be negatively charged and actually repel each other across the lanes causing the bands to get narrower as they progress down the gel.

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

Can I use CTAB rather than SDS in my sample buffer?

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 50 mM acetic acid and 50 mM 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).

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

Do you have recommended protocols for copper or zinc staining of NuPAGE gels?

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

Copper Stain: Staining solution - 0.3 M 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 0.3 M 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.

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

Is it okay to use protein gels past their expiration date?

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.


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Can I run your protein gels overnight?

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.

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

Do I need to increase the voltage when I run a 1.5 mm protein gel versus a 1.0 mm gel?

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

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

Why do you recommend running your protein gels at constant voltage?

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.

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

I am planning to run a NuPAGE Tris-Acetate gel to determine the molecular weight of a protein that supposedly has a high molecular weight. Do you offer an unstained standard for these gels?

For accurate estimation of molecular weight of high molecular weight proteins on NuPAGE Tris-Acetate gels with Tris-Acetate SDS buffer system, we recommend using the HiMark Unstained Protein Standard, Cat. No. LC5688.

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

I am planning to run a NuPAGE Tris-Acetate gel to resolve a 150 kDa protein. Which of your prestained protein standards should I use?

We recommend using the HiMark Prestained Protein Standard, Cat. No. LC5699 for determination of apparent molecular weight of high molecular weight proteins on NuPAGE Tris-Acetate gels with Tris-Acetate SDS buffer system. For accurate estimation of molecular weight of high molecular weight proteins on NuPAGE Tris-Acetate gels with Tris-Acetate SDS buffer system, we recommend using the HiMark Unstained Protein Standard, Cat. No. LC5688.

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

What are the storage conditions and shelf life for the HiMark Prestained Protein Standard?

We recommend storing the HiMark Prestained Protein Standard at -20 degrees C. The ladder is stable for 4 months when properly stored.

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

I ran my protein under native conditions on a Tris-Glycine gel. It has a pI that is higher than the pH of the Tris-Glycine transfer buffer. Can you offer some tips for transferring it?

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

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

What transfer conditions should I use for semi-dry transfer of NuPAGE Tris-Acetate gels using the Invitrogen Semi-Dry Blotter?

We recommend using 2X NuPAGE Transfer Buffer and performing the transfer at 20 V (constant) for 30-60 minutes.

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

My gel run is faster than normal with poor resolution. What could be causing this problem?

Here are possible causes and solutions:

- Buffers are too concentrated or incorrect. Check buffer recipe; dilute or re-make if necessary.
- Voltage, current or wattage is set at a higher limit. Decrease power conditions to recommended running conditions.

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

My gel will not run. Can you please help me troubleshoot?

It is important to determine whether the problem is with the power supply, the apparatus or the gel. Often, it helps to switch out the power supply or the lid to see if there is a faulty contact. Also, check to see whether the tape from the bottom of the gel cassette has been removed and whether the buffer core is damaged. Additionally, make sure there is sufficient buffer in the electrophoresis tank to cover the wells of the gel.

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

Which of your protein gels can I run using the Mini Gel Tank?

Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX), as well as our Invitrogen Mini gels and NuPAGE Mini gels can be run using the Mini Gel Tank. Please note that 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).

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

Which of your protein gels can I run using the XCell SureLock Mini-Cell?

Our New Bolt Bis-Tris Plus Mini gels (Cat. No. NWxxxxxBOX), as well as our Invitrogen Mini gels and NuPAGE Mini gels can be run using the XCell SureLock Mini-Cell.

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

After western detection, my membrane has a lot of spots. What could have gone wrong?

Here are possible causes and solutions:

- Membrane blotting pads are dirty or contaminated. Soak pads with detergent and rinse thoroughly with purified water before use. Replace pads when they become worn or discolored.
- Blocking was uneven. The incubation dish must be sufficiently big to allow thorough coverage of membrane. Shake or agitate during each step.

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

I am getting a lot of non-specific binding after western detection. Can you offer some tips?

Here are possible causes and solutions:

- Membrane contaminated by fingerprints or keratin proteins: Wear clean gloves at all times and use forceps when handling membranes. Always handle membranes around the edges.
- Concentrated secondary antibody used: Make sure the secondary antibody is diluted as recommended. If the background remains high, but with strong band intensity, decrease the concentration of the secondary antibody.
- Concentrated Primary antibody used: Decrease the concentration of the primary antibody.
- Affinity of the primary antibody for the protein standards: Check with the protein standard manufacturer for homologies with primary antibody.
- Insufficient removal of SDS or weakly bound proteins from membrane after blotting: Follow instructions for membrane preparation before immunodetection.
- Short blocking time or long washing time: Make sure that each step is performed for the specified amount of time.

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

I am getting very high background after western detection. Can you please offer some tips?

Here are possible causes and solutions:

- Insufficient blocking or non-specific binding: We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050).
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Blot is overdeveloped: Follow recommended developing time and remove blot from substrate when signal - to -noise ratio is acceptable.
- Insufficient washing ; Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentrated secondary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.
- Concentrated primary antibody used: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

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

I am unable to visualize my protein bands after western detection. What is the problem?

Here are possible causes and solutions:

- The primary antibody and secondary antibody are not compatible: Use a secondary antibody that was raised against the species in which the primary antibody was raised.
- The primary antibody is too dilute: 1) Use a more concentrated antibody solution. 2) Incubate longer (e.g., overnight) at 4 degrees C. 3) Use fresh antibody and keep in mind that each time an antibody solution is used, its effective antibody concentration decreases.
- Something in your blocking buffer interferes with binding of the primary and/or secondary antibody: Try an alternate blocking buffer ± a mild surfactant like Tween-20 (0.01-0.05% v/v). There are many blocking buffer recipes available, based on non-fat dry milk, BSA, normal serum, gelatin and mixtures of these and other materials. Note that BSA (1-5%) is considered the best blocker for nitrocellulose membranes. It is easy to check the efficacy of different blocking buffers by performing dot-blots.
- The primary antibody does not recognize the protein in the species being tested: 1) Evaluate primary antibodies by dot-blotting first to how well they react with your protein. 2) Check the immunogen sequence, if provided, and determine if it is found in your protein. 3) If no immunogen sequence is available, perform a PubMed/BLAST alignment to assess the degree of homology between your target protein and the protein against which the antibody was generated. Note that many antibodies against human proteins will also recognize the non-human primate version because there is usually a high degree of amino acid identity. In contrast, many antibodies against human proteins will not recognize the corresponding proteins from rodents (and vice versa). Remember that significant homology between sequences does not guarantee that the antibody will recognize your protein. 4) Always run the recommended positive control, if available.
- Insufficient protein is bound to the membrane or the protein of interest is not abundant enough in the sample: 1) Load at least 20-30 ?g protein per lane on your gels (as a starting point), since proteins representing less than ~0.2% of the total protein are difficult to detect on western blots. 2) Use an enrichment step to increase the concentration of the target protein. For example, prepare two nuclear lysates prior to blotting nuclear proteins or perform an immunoprecipitation (IP) prior to SDS-PAGE. 3) Reduce the volume of cell extraction buffer used to lyse your cells or tissue. 4) Be sure to use freshly prepared protease inhibitors and phosphatase inhibitors, if needed, in your protein extraction buffer. 5) Run the recommended positive control, if available.
- Poor or no transfer of the proteins to the membrane 1) Check the protein transfer efficiency with a reversible protein stain like Invitrogen Reversible Membrane Protein Stain, ponceau S, amido black or use pre-stained molecular weight standards. 2) Verify that the transfer was performed with the correct electrical polarity. 3) Remember that proteins with basic pI values (e.g., histones) and high MW may not transfer well. 4) Remember that if your target protein has a low MW (≤10 kDa), it may transfer more quickly than expected. 5) If you are using PVDF membranes, make sure to pre-soak the membrane in methanol first before soaking it in transfer buffer. Note that methanol in transfer buffer increases protein binding to nitrocellulose, but omitting methanol can increase transfer efficiency of high MW proteins. 6) Low MW proteins may pass through the 0.45 µm pores in nitrocellulose membranes, so switch to NC with 0.2 or 0.1 µm pores instead.
- Excessive washing or blocking of the membrane:- 1) Avoid over-washing the membrane. Extra washing will not allow you to visualize your protein of interest if there are other problems with your blot. 2) Avoid over-blocking by using high concentrations of the blocking buffer components or long incubation times. Too much blocking can prevent your antibodies from binding to your protein. Gelatin, in particular, can mask proteins on the blot, so avoid it, if possible. Milk can also mask proteins, so instead of using 5% milk in your blocking buffer, try using it at 0.5% instead, or remove it altogether. 3) Switch to a different blocking reagent and/or block the blot for less time.
- Using the same solution of diluted primary antibody repeatedly: Use freshly-diluted antibody for each western blot because the effective concentration of a diluted antibody decreases each time it is re-used. Also, remember that dilute solutions of antibodies are less stable and may lose their activity rapidly.
- The enzyme conjugated to your secondary antibody is not working: 1) Make a fresh dilution of your secondary antibody conjugate each time you need it. Enzymes (and antibodies) may lose activity quickly in dilute solutions. 2) Omit sodium azide in buffers if you are using HRP-conjugated antibodies. 3) Avoid high heme concentrations (from blood contamination), which can interfere with HRP-based detection. 4) Avoid using phosphate in buffers with alkaline phosphatase-antibody conjugates because phosphate inhibits enzyme activity.
- Your colorimetric or other detection reagent is old and inactive: 1) Use fresh enzyme substrate for each experiment. 2) Don't use ready-to-use substrate reagents if they have changed color on their own or if they have passed their expiration date. 3) Do not dilute substrate solutions unless instructed to do so in the product manual.

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

I ran my protein sample on one of your gels and the bands look non-distinct and smeary after western detection. What should I do?

Here are some suggestions:

- Make sure that the correct amount of protein is loaded per lane; loading too much protein can cause smearing.
- Bands will not be as well resolved in low percentage gels; try using a higher percentage gel.
- This may be due to the antibody being too concentrated. We recommend following the manufacturer's recommended dilution or determining the optimal antibody concentration

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

I ran my protein samples on one of your protein gels and saw V-shaped protein bands. Can you please help me troubleshoot?

V-shaped protein bands are caused by the presence of DNA in the sample. The artifact might be eliminated or minimized by shearing the DNA with additional sonication after the SDS-solubilization step. Alternatively, the DNA can be removed from the sample using an ultra-centrifuge.

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

Can I use the NativePAGE Sample and Running buffers with Invitrogen Tris-Glycine gels or NuPAGE Tris-Acetate gels for native applications?

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 or Invitrogen Tris-Glycine gels. For those gels, we recommend using the Invitrogen Tris-Glycine Native Sample and running buffers.

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

I have set up my NuPAGE gel to run and have switched on the power supply, but my gel is not running and there is no voltage or current reading on the power supply. What is wrong?

*Double check that the tape on the bottom of the gel has been removed.
*Make sure that the gel(s) are oriented so that the taller sides of the cassette (with the printing) are facing the outside of the electrophoresis unit.
*Make sure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer. If the wells are not covered, check for leaks and reseal.
*Double check to see if there are any loose electrodes or connections on the Mini cell unit.
*Check the power supply unit.

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

I am running my NuPAGE gel using the XCell SureLock Mini Cell but the leads do not fit into my power supply. Can you please help?

You may purchase the ZOOM adapters, Cat. No. ZA10001 to help you connect your leads to the power supply.

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

I am trying to load my samples on a NuPAGE gel but am not able to see the sample wells. Can you please suggest some tips?

We recommend marking the cassette at the bottom of the wells with a marker pen prior to assembling the upper buffer chamber. Also, we recommend illuminating the bench area with a light source placed directly behind the XCell SureLock unit.

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

I am running my NuPAGE gel using the XCell SureLock Mini Cell, and the gel run is taking longer than usual. What could be causing this?

Here are possible causes and solutions:

1) Buffers are too dilute: Check buffer recipe; remake if necessary.
2) Upper buffer chamber is leaking: Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
3) Voltage is set too low: Set correct voltage.

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

I had problems transferring my larger molecular weight proteins from my NuPAGE gel. Can you please offer some suggestions?

For proteins larger than 100 kDa, we recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing methanol and 0.01% SDS.

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

The protein bands in some of my gel lanes are irregular or wavy? What would have caused this problem?

This could be due to:

*Debris in the well
*High salt in the sample (make sure that the salt concentration does not exceed 50-100 mM)
*Running buffer issue
*Gel casting error

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

I am seeing a very wavy and uneven dye front with my samples. Can you please help me troubleshoot?

This could be due to a gel polymerization issue combined with incorrect sample preparation (final sample dilution less than 1X). Please try a different lot of the same gel and make sure that the sample is correctly prepared.

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

I am seeing a faint, artifact doublet band at ~60 kDa in all my lanes. This band seems to be getting darker the longer I stain the gel. What could be causing this?

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.

I loaded different protein samples in each well but I see the same protein band in several neighboring lanes. What could have happened?

Possible cause:

*Carry-over contamination of sample from one well into neighboring wells due to loading error
*Contaminated running buffer
*Gel casting error: malformed wells

Remedy:

*Use a gel loading tip to load wells
*Reduce the sample volume
*Do not delay while loading wells
*Do not delay after the run, as proteins can diffuse horizontally; a full well left next to an empty well would eventually contaminate the empty well over time.

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

My protein bands appear to be skewed or distorted. What is the problem?

Possible cause:

*Poor polymerization around sample wells
*High salt concentration in sample
*Uneven gel interface
*Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly
*Uneven heating of the gel
*Insoluble material in the gel or inconsistent pore size throughout the gel
*Air bubble during the run

Remedy:

*Remove excess salt/other material by dialysis, Sephadex G-25 or any other desalting column or using an Amicon concentrator.
*Either use a cooled apparatus or reduce the current at which electrophoresis is performed.

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

I ran my reduced protein samples under denaturing conditions and am seeing doublet protein bands when I expect to see single bands. Why is this happening?

A portion of the protein sample may have re-oxidized during the run, or may not have been fully reduced prior to the run. We recommend preparing fresh sample solution using fresh beta-mercaptoethanol or dithiothreitol (DTT). For NuPAGE gels, we recommend adding antioxidant to the running buffer.

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

My gel seems to be lifting off the cassette. What could be causing this?

Gel lifting off the cassette can be caused by:

*Expired gels that are degrading
*Improper storage of gels
*Too much heat accumulating during the electrophoresis run due to excessive current
*Insufficient polymerization of the polyacrylamide

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

I am seeing a faint shadow, or "ghost" band below a normal and expected protein band? What could be the potential issue?

Ghost bands are usually attributed to a slight lifting of the gel from the cassette, which results in the trickling down of some sample beyond its normal migration point. It then accumulates and appears as a faint second band.

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

My protein bands in the outer lanes of the gel show a "smiling" effect. Can you please help me troubleshoot?

"Smiling" bands may be the result of the acrylamide in the gel breaking down, leaving less of a matrix for the proteins to migrate. We recommend checking to ensure that the gels have not been used past their expiration date.

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

I see dumbbell or barbell shaped bands after protein electrophoresis. What could be causing this?

Barbell shaped bands are a result of loading too large of a sample volume. When a large sample volume is loaded, part of the sample tends to diffuse to the sides of the wells. When the run begins and the sample moves through the stacking portion of the gel, the sample will incompletely stack causing a slight retardation of the portion of the sample that diffused to the sides of the wells. This effect may be intensified for larger proteins, whose migration is more impeded in the low concentration acrylamide of the stacking gel. To alleviate the problem, we recommend concentrating the protein and loading a smaller volume. This gives a "thinner" starting zone.

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

Why do I get streaking forward or "frowning" of one of my samples on my protein gel?

Here are possible causes and solutions:

1) Sample overload: Do not overload samples
2) Addition of reducing agent that is not fresh: Reduce samples right before loading and do not use samples that have been stored in reducing agent
3)Re-oxidation of the protein during the run: Add antioxidant to the running buffer if you are running NuPAGE gels
4) Presence of highly hydrophobic regions where the protein can exclude SDS: Load the sample with 2X sample buffer instead of 1X sample buffer
5) Excess salt in the sample: Precipitate and reconstitute in lower salt buffer
6) Not enough SDS in the sample: Add SDS to the upper buffer chamber (try 0.1%, 0.2%, 0.3% and 0.4% SDS)

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

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.

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

Can I use the NativePAGE Sample and Running buffers with NuPAGE Tris-Acetate gels for native applications?

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. When running Tris-Acetate gels under native conditions, we recommend using the Invitrogen Tris-Glycine Native Sample and running buffers.

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

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.

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

What stains do you recommend for NuPAGE gels?

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.

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

What are the main advantages of NuPAGE gels over Invitrogen Tris-Glycine gels?

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 Invitrogen 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 Invitrogen 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

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

How does the operating pH for Tris-Glycine gels differ from that for NuPAGE Bis Tris and NuPAGE Tris-Acetate gels?

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.

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.

How should I store my NuPAGE Tris-Acetate gels?

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

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

Can I run my NuPAGE Tris-Acetate gels under native and denaturing conditions?

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 Invitrogen Tris-Glycine Native Running buffer and Invitrogen Tris-Glycine native sample buffer.

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

Can I run Mini gels with 10 cm gel cassettes using a Bolt Mini Gel Tank?

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 (https://tools.thermofisher.com/content/sfs/manuals/mini_gel_tank_man.pdf).

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 (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gel-electrophoresis-chamber-systems/mini-gel-tank/resources-upgrading-bolt-mini-gel-tank.html) or in this video (https://www.youtube.com/watch?v=1FtiX8Skllw).

Additional resources can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gel-electrophoresis-chamber-systems/mini-gel-tank/resources-upgrading-bolt-mini-gel-tank.html).

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

How do you recommend transferring Midi gels?

Midi gels can be transferred using:

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

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

Will NP-40 affect the migration of my protein samples?

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.

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

Do your Invitrogen protein gels contain any carbohydrates and are they suitable for carbohydrate analysis?

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

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

What is the material used for making your Invitrogen precast gel plastic cassettes?

The cassettes are made of a styrene copolymer.

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

Can I recycle your Invitrogen precast gel plastic cassettes?

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.

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

What is the difference between Invitrogen Mini and Midi gel formats?

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.

Midi gels:
*NuPAGE Bis-Tris, NuPAGE Tris-Acetate, & Invitrogen Tris-Glycine: Gel dimensions are 13cm x 8.3cm and Cassette dimensions are 15cm x 10.3cm.

Mini gels:
*NuPAGE Bis-Tris, NuPAGE Tris-Acetate, & Invitrogen Tris-Glycine: Gel dimensions are 8cm x 8cm and Cassette dimensions are 10cm x 10cm.
*New Bolt Bis-Tris Plus (Cat. No. NWxxxxxBOX): Gel dimensions are 8cm x 8.3cm and Cassette Dimensions are 10cm x10cm.
*Original Bolt Bis-Tris Plus (Cat. No. BGxxxxxBOX): Gel dimensions are 8cm x 8.3cm and Cassette Dimensions are 10cm x 10.5cm.

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

What are the dimensions of your precast protein gels?

All of our Invitrogen precast protein gels (NuPAGE gels, Bolt Bis-Tris Plus gels, and Novex gels) are available in Mini format. Our Mini gel dimensions are 8 cm x 8 cm and the cassette dimensions are 10 cm x 10 cm.

Our NuPAGE Bis-Tris, NuPAGE Tris-Acetate, and Novex Tris-Glycine Plus gels are also available in the wider Midi format. Our Midi gel dimensions are 8 cm x 13 cm and the cassette dimensions are 10 cm x 15 cm.

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

Are your precast protein gels available in Mini and Midi formats?

All our Invitrogen protein gels are available in Mini format. Certain gel chemistries (NuPAGE Bis-Tris, NuPAGE Tris-Acetate, and Invitrogen Tris-Glycine gels) are also available in the wide Midi format.

Note that Bolt Bis-Tris gels are not available in the Midi format and our Thermo Scientific Precise precast gels are only available in Mini format.

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

When running two protein gels, do I need to double the voltage?

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.

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

Which protein standard do you recommend using with gels run under native conditions?

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

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

Can I run reduced and non-reduced protein samples on the same gel?

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.

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

Can I store my reduced protein samples for later use?

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.

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

What is the ratio of acrylamide:bisacrylamide and percentage of cross-linker in your Invitrogen precast gels?

*Tris-Glycine gels (except 4% Tris-Glycine gels) have a 34.5:1 Acrylamide:bisacrylamide and 2.6% Crosslinker.

*4% Tris-Glycine gels have a 76:1 ratio Acrylamide:bisacrylamide and 1.3% Crosslinker.

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

What is the percentage of the stacking gel in your Invitrogen precast protein gels?

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.

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

Do your Invitrogen precast protein gels contain a stacking gel?

Our Invitrogen 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.

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

What are the recommended sample loading volumes and protein loading amounts for your precast protein gels?

*Tris-Glycine and Invitrogen Tricine Mini gels: see here (http://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf), Page 8

*NuPAGE Tris-Acetate and NuPAGE Bis-Tris Mini gels: see here (http://tools.thermofisher.com/content/sfs/manuals/nupage_tech_man.pdf), Page 10

*Bolt Bis-Tris Plus Mini gels: see here (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/bolt-bis-tris-gels.html)

*Thermo Scientific Precise Tris-HEPES gels: see here (https://tools.thermofisher.com/content/sfs/manuals/MAN0011499_Precise_Protein_Gels_UG.pdf), Page 1

*Midi gels (Invitrogen Tris-Glycine, NuPAGE Bis-Tris and NuPAGE Tris-Acetate): see here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/novex_midigel_man.pdf), Page 4

*Thermo Scientific Precise Tris-Glycine gels: see here (https://tools.thermofisher.com/content/sfs/manuals/D25MAN0011814_Precise_TrisGlycine_Gels_UG.pdf), Page 1

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

Do your precast protein gels contain SDS?

Our precast protein gels do not contain SDS but they can be run under denaturing conditions when used with the appropriate denaturing 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.

*Invitrogen Tris-Glycine gels: For Native electrophoresis, use Invitrogen Tris-Glycine Native Running Buffer. For Denaturing electrophoresis, use Invitrogen Tris-Glycine SDS Running Buffer

*NuPAGE Tris-Acetate gels: For Native electrophoresis, use Invitrogen Tris-Glycine Native Running Buffer. For Denaturing electrophoresis, use NuPAGE Tris-Acetate SDS Running Buffer

*NuPAGE Bis-Tris gels: For Denaturing electrophoresis, use NuPAGE MOPS-SDS Running Buffer or NuPAGE MES-SDS Running Buffer

*Bolt Bis-Tris Plus gels: For Denaturing electrophoresis, use Bolt MOPS SDS Running Buffer or Bolt MES SDS Running Buffer

*Thermo Scientific Precise Tris-Glycine gels: For Native electrophoresis, use Tris-Glycine SDS Running Buffer without SDS added. For Denaturing electrophoresis, use Tris-Glycine SDS Running Buffer.

*Thermo Scientific Precise Tris-HEPES gels: For Denaturing electrophoresis, use Tris-HEPES SDS Running Buffer.

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