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View additional product information for NativePAGE™ Bis-Tris Mini Protein Gels, 4 to 16%, 1.0 mm - FAQs (BN1002BOX, BN1004BOX)
36 product FAQs found
The NativePAGE Invitrogen Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analyzing native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessing purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow.
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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.
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Yes, NativePAGE gels are compatible with our Mini Gel tank, however there is a small variation from the original protocol.
The following protocol are for using NativePage gels with the Mini Gel Tank depending on if you are also performing a western transfer or not:
If you are NOT performing a western transfer:
1. Prepare 250 mL of each buffer (Anode and Dark Blue Cathode) per gel
2. After inserting a loaded NativePAGE gel into the Mini Gel Tank and pulling the clamp forward, fill the front cathode buffer chamber to the Fill Line with Dark Blue Cathode Buffer (~200 mL per gel)
3. Add 220 mL of Anode Buffer to the back anode buffer chamber for that gel. Start the gel run
If you are performing a western transfer:
1. Prepare 250 mL of Dark Blue Cathode Buffer, 250 mL of Light Blue Cathode Buffer, and 500 mL of Anode Buffer per gel
2. After inserting a loaded NativePAGE gel into the Mini Gel Tank and pulling the clamp forward, fill the front cathode buffer chamber to the Fill Line with Dark Blue Cathode Buffer (~200 mL per gel
3. Add 220 mL of Anode Buffer to the back anode buffer chamber for that gel
4. Start the gel run; pause the run after the dark blue dye has run ~1/3 of the way through gel
a. Pour out the buffers from the Mini Gel Tank
b. Refill the back anode buffer chamber with 220 mL of Anode Buffer per gel
c. Fill the front cathode buffer chamber to the Fill Line with Light Blue Cathode Buffer (~200 mL per gel)
5. Resume the gel run
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We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725 for native gel electrophoresis with Tris-Glycine, NuPAGE Tris-Acetate or NativePAGE gels.
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During NativePAGE runs, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.
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You can try the standard recommendation for cleaning which would be washing the unit with a mild detergent and rinsing with deionized water.
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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.
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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.
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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.
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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
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During NativePAGE electrophoresis, it is common for the current to drop below 1 mA. Most power supplies register this as a “No Load” error and automatically shut off, resulting in the stopping of the gel run. This can be bypassed in some power supplies by disabling or turning off the “Load Check” feature.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Although the gels share some major chemical components, they are not interchangeable. NuPAGE Bis-Tris gels are optimized for denaturing and reducing conditions, and their neutral pH makes it difficult to use them for native applications as most proteins will have no charge or positive charge. Therefore, native applications with these gels are not recommended. NativePAGE Bis-Tris gels on the other hand have a different formulation that has been optimized for native electrophoresis with highest resolution. The buffers for the two types of gels should not be interchanged.
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Yes, please take a look at the Western Blotting NativePAGE Invitrogen Bis-Tris Gels Using the iBlot 7-Minute Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.18870.file.dat/native-with-iblot-app-note-v3.pdf).
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We recommend using the NuPAGE Transfer buffer for blotting of NativePAGE gels. PVDF is the recommended blotting membrane and works well in terms of transfer and detection. Nitrocellulose is not compatible with blotting of NativePAGE gels, as the nitrocellulose membrane binds the Coomassie G-250 dye very tightly and is not compatible with alcohol-containing solutions needed to destain the membrane and fix the proteins.
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NativePAGE Invitrogen Bis-Tris Gels are compatible with most of the standard Coomassie R-250 or G-250 staining formulations. The Invitrogen Colloidal Blue Staining Kit (Cat. No. LC6025) is recommended for the most sensitive Coomassie staining in NativePAGE Gels. SimplyBlue Safe Stain (Cat. No. LC6060) is not recommended for use with NativePAGE gels, due to special fixing requirements. Both the SilverQuest Silver Staining Kit (Cat. No. LC6070) and SilverXpress Silver Staining Kit (Cat. No. LC6100) are suitable for staining of NativePAGE Gels. However, for best overall results and lower background, we recommend the SilverQuest Kit as the best choice.
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No, most of our protein standards (including the Invitrogen Sharp prestained and unstained standards) are already denatured and reduced, and will not resolve well in a native gel. For NativePAGE electrophoresis, we offer the NativeMARK Unstained Protein Standard (Cat. No. LC0725). It is a ready-to-use protein marker that allows for molecular weight estimation of proteins using native gel electrophoresis.
Please note that even with a native standard, molecular weight estimation in native electrophoresis is very inaccurate, as gel migration can be significantly affected by differing charge and conformation of individual proteins. For more accurate estimations, use SDS-PAGE separation or mass spectrometry analysis.
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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.
NativePAGE gels have been successfully used to perform EMSA showing protein-protein interactions between two purified proteins, but we have not tested NativePAGE gels for EMSA analysis of interactions between nucleic acids (either DNA or RNA) and a protein or protein complex.
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Samples requiring non-ionic detergents for solubility are not compatible with traditional native Tris-Glycine PAGE because as the proteins migrate into the polyacrylamide gel, they leave behind the non-ionic detergents. In the absence of non-ionic detergent, the proteins aggregate and form vertical streaks at the top of the lane. When using blue native electrophoresis (NativePAGE gels), the Coomassie G-250 dramatically reduces aggregation to allow the resolution of membrane protein complexes not seen in the Tris-Glycine gel.
In addition, compared to the operative pH of the Tris-glycine system (pH 9.3-9.5), the lower operative pH of the NativePAGE gels (pH 7.5-7.7) may help to retain the native structure and/or activity of proteins sensitive to alkaline pH.
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The NativePAGE Invitrogen Bis-Tris Gels have a wide range of separation throughout the low- and high- molecular weight ranges:
*The NativePAGE Invitrogen 3-12% Bis-Tris Gels resolve proteins in the molecular weight range of 30-10,000 kDa.
*The NativePAGE Invitrogen 4-16% Bis-Tris Gels resolve proteins in the molecular weight range of 15-10,000 kDa.
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We recommend storing them at 4-25 degrees C. They should not be frozen.
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The recommended sample loading volumes and protein loading amounts for the NativePAGE well formats are listed on Page 3 of the NativePAGE Invitrogen Bis-Tris Gel System manual (http://tools.thermofisher.com/content/sfs/manuals/nativepage_man.pdf).
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Our NativePAGE gels do have a stacking gel. The stacking gel for these gels is a ~1 cm region at the top of the gel where the acrylamide percentage is low (4%) and constant. Below the stacking gel, the acrylamide percentage begins to increase in the gradient portion of the gel. However, the gel buffer is the same throughout the gel. So the stacking gel in NativePAGE gels is not the same as in the Laemmli system where the stacking gel has a different pH causing a decreased ion mobility for the trailing ions. Also the entire NativePAGE gel is cast in one continuous delivery due to which no demarcation line is seen between the resolving (or gradient) portion of the gel and the stacking gel.
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The NativePAGE 5% G-250 Sample Additive is a concentrated stock solution of Coomassie G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE gel electrophoresis. Native proteins normally migrate on the gel according to natural charge/pI in the pH of gel buffer, but adding the Coomassie G-250 renders an associated negative charge even to high pI proteins that would normally have a positive charge. It binds to proteins non-specifically without denaturing them. The NativePAGE 5% G-250 Sample Additive is added to the cathode buffer and, optionally, to samples as well.
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The NativePAGE 5% G-250 Sample Additive (Cat. No. BN2004) is a concentrated stock solution of Coomassie G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE gel electrophoresis. The exact composition of the NativePAGE 5% G-250 Sample Additive is proprietary. It does not contain a detergent.
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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
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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.
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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.
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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.
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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.
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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
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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.
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"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.
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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.
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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)
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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.