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View additional product information for Novex™ Tris-Glycine Mini Protein Gel, 4 to 20%, 1.0 mm, 1-well - FAQs (EC6021BOX)
62 product FAQs found
The length of the well in the Invitrogen 1-well gels is 7.6 cm or 2.956 inches.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
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.
Gel straightness is defined as the straightness across all lanes of the gel, measured at the bottom, expressed relative to the total length of the gel. For example, a gel with straightness of 0.020 Rf is flat to within 2% of the length of the gel (1.6 mm) across. Band curvature is defined as the curvature of the bands in the outer lanes of the gel, expressed relative to the total length of the gel. For example, bands with curvature of 0.010 Rf are straight to within 1% of the length of the gel (0.8 mm).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The QC of our gels includes several processes:
1) Each gel is checked by eye for visible anomalies.
2) Under defined conditions, gels retained from each lot are tested as follows:
--When gels are run at a defined voltage, the resulting current and power of the electrophoresis are measured.
--Protein samples are electrophoresed on test gels to determine the gel run time and the protein band quality after electrophoresis. Bands are examined for: straightness within bands, curvature of bands across the gel ("smiling" or "frowning"), and reproducibility of the Rf values for protein molecular weight markers. According to these results, a Certificate of Analysis is created, which is available upon request.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here is the transfer protocol optimized for the Bio-Rad Semi-Dry Transfer Unit. NuPAGE transfer buffer can be used for transfer of Tris-Glycine gels.
- Working transfer buffer: 10% methanol, 1:1,000 Antioxidant in 2X NuPAGE transfer buffer (Bis-Tris 50 mM and Bicine 50 mM). If you need to prepare 100 mL of the working buffer from the NuPAGE 20X Transfer Buffer (Cat. No. NP0006), mix the following: 10 mL of 20X transfer buffer, 10 mL of MeOH, 100 µL of Antioxidant, 80 mL of DI H2O.
- Filter papers: The transfer buffer-soaked filter papers of the sandwich are the only reservoir in the Semi-Dry Transfer Cell. If Invitrogen pre-cut membrane/filter sandwiches are used, at least 2 extra filter papers (0.4 mm/filter in thickness) on each side of the gel (or membrane) are required. When assembling one gel/membrane sandwich, presoak 6 Invitrogen filter papers (or 2 thicker filter papers) and 1 membrane in working transfer buffer (prepared in step 1) and sandwich them on the top of the anode plate as follows: filter paper--filter paper--filter paper--membrane--gel--filter paper--filter paper--filter paper
- Blotting conditions: We found 15 V for 15-30 min is optimal for NuPAGE transfer buffer in the Bio-Rad Semi-Dry Transfer Cell. Semi-dry transfer units from other manufacturers should be used according to unit's instructions.
- For transfer of large proteins (100 kDa or larger), pre-equilibrate the gel with 0.02-0.04% SDS in 2X transfer buffer without methanol for 10 min before assembling the sandwich. Please note that transferring Tris-Glycine gels using NuPAGE transfer buffer in the Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell may be less efficient than using Tris-Glycine transfer buffer (Cat. No. LC3675) in the XCell II Blot Module (semi-wet).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, we recommend using 2X Tris-Glycine Transfer Buffer containing 10% methanol and performing the transfer at 20 V (constant) for 30-60 minutes.
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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.
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.
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.
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.
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 Invitrogen 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, Cassette Clamp Cam Handle Set). Instructions for replacement of the cam handles 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).
Our Midi gels can be run using the XCell4 SureLock Midi-Cell.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
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.
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.
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.
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.
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.
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.
Background is generally higher in gels with less than 10% acrylamide percentage due to penetration and trapping of colloids within the large pores of these gels. Excess background may be reduced by incubating the gel in 25% methanol solution until a clear background is obtained. Be aware that the dye will also be partially removed from the bands and that prolonged incubation in >25% methanol will result in complete destaining of protein bands and background.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
It may be possible to reduce background by using the protocol provided for NuPAGE Invitrogen Bis-Tris gels/Small Peptides. This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background. The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE protocol.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
*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.
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.
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.
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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 volatage
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
This can happen if Tris-Glycine gels are run using NuPAGE Running buffer containing Antioxidant. Please make sure that the correct Tris-Glycine Running buffer is used with Tris-Glycine gels.
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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
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
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.
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.
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.
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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Tris-Glycine gels may be transferred in CAPS buffer (10mM CAPS (3-cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0.
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Tris-Glycine gels are compatible with any of the standard Coomassie staining procedures. They are also compatible with most silver staining protocols.
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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 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.
Our Tris-Glycine gels do not contain SDS. They can be run under native conditions by using the Invitrogen Tris-Glycine Native Sample buffer and the Invitrogen Tris-Glycine Native Running buffer. They can be run under denaturing conditions by using the Invitrogen Tris-Glycine SDS Sample buffer and the Invitrogen Tris-Glycine SDS Running buffer.
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Invitrogen 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. Invitrogen Tris-Glycine Gels provide reproducible separation of a wide range of proteins into well resolved bands.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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
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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.
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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.
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.
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.
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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.
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The cassettes are made of a styrene copolymer.
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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.
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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.
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.
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.
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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.
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We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725.
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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.
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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.
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*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.
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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.
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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.
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*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.
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.