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View additional product information for Novex™ IEF Protein Gels, pH 3-7, 1.0 mm - FAQs (EC6645BOX, EC66452BOX)
62 product FAQs found
There are several reasons why streaking may occur.
(1) Sample is not completely solubilized prior to application.
(2) Sample is poorly soluble in rehydration solution.
(3) Non-protein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2-D result, particularly toward the acidic side of the gel.
(4) Ionic impurities are present in sample.
(5) Ionic detergent is present in sample.
(6) Sample load is too high.
(7) Underfocusing. Focusing time was not long enough to achieve steady state focusing.
(8) Overfocusing. Extended focusing times (over 100,000 Vh) may result in electroendosmotic water and protein movement, which can produce horizontal smearing.
What should be done?
(1) Be sure that the sample is completely and stably solubilized. Note: Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.
(2) Increase the concentration of the solubilizing components in the rehydration solution.
(3) Modify sample preparation to limit these contaminants or dialyze protein.
(4) Reduce salt concentration to below 10 mM by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides and other small molecules.
Note: Specific and non-specific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples. If the sample preparation cannot be modified, the effect of ionic impurities can be reduced by modifying the IEF protocol. Limit the voltage to 100-150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip.
(5) If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the non-ionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins.
(6) Extend focusing time. Load less sample.
(7) Prolong focusing time.
(8) Reduce focusing time.
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It is not unusual for antibodies (even monoclonals) to be differentially glycosylated, and therefore not focus well on IEF gels. Sometimes a monoclonal antibody can even run more smeary or unfocused than a polyclonal, over a range of pH 6.5 to 8.0. Try running the gel longer to improve the focus, but often the improvement is minimal if anything.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The initial voltage is to set up the ampholytes in a pH gradient, the second step actually drives the proteins to their pI. The third step is to "fine focus" or sharpen the protein bands.
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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. All detergents and 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 nonionic detergents significantly interfere with SDS-PAGE.
We recommend that you keep the ratio of SDS to lipid or other detergent at 10:1 (or greater) to minimize these effects.
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Yes, the fix serves two purposes: it fixes the sample in the IEF gel and it helps to remove gel background.
If you do not use the fixing solution, the background on the gels will be high and detection will be less sensitive.
High background is caused by ampholytes remaining in the gel.
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No, we do not recommend using the Invitrogen IEF gels for reverse IEF.
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1) If the protein has a pI greater that 8.5, it may be able to be resolved on a pH 3-10 gel.
2) The protein may be insoluble.
3) There may be protein loss if the fixative is too weak in the fixation step prior to staining.
4) Your sample load may be too low.
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No, the SDS gives the protein complex a strong negative charge and therefore an unreliable separation based on isoelectric point. If you have a protein that is moderately soluble, then you can try to solubilize the protein in a nonionic detergent (between 0.1-0.5%) such as Triton X-100, NP-40, or Tween 20. The nonionic detergent may help to solubilize the protein, however, this needs to be tested empirically, because the characteristics of the protein such as its purity, concentration, and ionic strength will influence how well it will run on the gel. Very hydrophobic proteins, like membrane proteins, will not work well, even if a nonionic detergent is added to the sample buffer.
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Yes, you can use just arginine (either form of arginine) as long as the arginine free base form is used and not the hydrochloric form. Note that the lysine used to make the mixed buffer should be free base lysine; free acid lysine should not be used as a substitute. The reason for adding basic amino acids to the cathode buffer is to increase the ionic strength of the buffer in order to fortify the basic end.
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The length of the 2D well is 6.5 cm. The IEF gel strip must be trimmed down to 5.8-5.9 cm before positioning it into the 2D well, even after it has been soaked in methanol to shrink the gel. For the 7 cm IPG strips, significant trimming would be necessary if the gels with the 2D wells are to be used. For IPG strips, the longer IPG well (found in the ZOOM gels) is recommended.
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For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer:
1) After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 min.
2) Chill the 0.7% acetic acid that will be used as the transfer buffer.
3) Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (-). This is OPPOSITE from the typical western blot with SDS-PAGE, where the negatively-charged protein will migrate toward the anode (+) side during the transfer.
4) Transfer for one hour at 10 V.
Tip for handling the IEF gel: the 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.
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First of all, the standards should be examined for proper focusing. A second likely cause is a mixing of the anode and cathode buffers. Even a small amount can cause aberrations. Unlike Tris-Glycine or Tricine gels, where the inner and outer buffer chambers are filled with the same buffer, extreme caution must be taken to prevent splash-over in IEF gels. High salt concentration can also cause bands to run very unevenly. Additionally, high salt in a sample lane can affect the run of adjacent standard lanes with low salt.
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It is recommended that an IEF gel be run at constant voltage (V): 100 V for the first hour, 200 V for the second hour, and 500 V for the last half hour. Since the voltage will increase over time if the run is set at constant current, set the run at 5 mA per gel and set a voltage limit of 500 V.
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The bubbling problem is probably carbon dioxide outgassing from the gel. Carbon dioxide is a weak acid and will migrate into the acidic region of the IEF gel and come out of solution as it concentrates. Thorough degassing of the cathode buffer will reduce the problem. In addition, electrophoresis can be carried out in the cold room so that the carbon dioxide will not come out of solution. If some bubbles form in the gel in the last few minutes of the run, they are unlikely to cause visible distortion. If they form earlier in the run, they may cause distortion because they act as insulators and change the gel temperature. Bubbles typically form between the gel surface and the cassette.
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IEF gels contain 5% acrylamide, 2.6% bisacrylamide, and 2% ampholytes.
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One liter: 114.6 g TCA (trichloroacetic acid), 34.6 g sulfosalicylic acid (electrophoresis grade, Fisher), 850.8 mL deionized water.
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The nomenclature of the vertical IEF gels refers to the pI range of ampholytes that are added to the IEF gels, not necessarily the focusing performance range. In practice, a 3-10 IEF gel can only resolve proteins with a pI of up to about 8.5. This is due to "cathodic drift". During electrophoresis, acrylamide hydrolyzes to polyacrylic acid. This happens to a greater extent at higher pH than at lower pH. Consequently, the lower pH acid groups formed at the high pH end of the gradient titrate out the basic groups, lowering the pH gradient at the basic end of the gel to about 8.5. Basic proteins with a pI of 8.5 to 10.0 may migrate into the gel initially, but will migrate backwards out of the gel as the run proceeds, and even if the proteins were focused at the pIs outside of the performance range, they will not hold.
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During IEF, 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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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:
- 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.
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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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It is not unusual for antibodies (even monoclonals) to be differentially glycosylated, and therefore not focus well on IEF gels. Sometimes a monoclonal can even run more smeary or unfocused than a polyclonal, over a range of pH 6.5 to 8.0. Try running the gel longer to improve the focus, but often the improvement may be minimal if any at all.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
*The standards should be examined for proper focusing.
*A likely cause is a mixing of the anode and cathode buffers. Even a small amount can cause improper runs. Unlike Tris-Glycine or Tricine gels, where the inner and outer buffer chambers are filled with the same buffer, extreme caution must be taken to prevent splash-over.
*High salt concentration can cause bands to run very crookedly. Also high salt in a sample lane can affect the run of adjacent standard lanes with low salt.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The bubbles are probably caused by carbon dioxide outgassing from the gel. Carbon dioxide is a weak acid and will migrate into the acidic region of the IEF gel and come out of solution as it concentrates. Thorough degassing of the cathode buffer will reduce the problem. In addition, the gel can be run in the cold room so that the carbon dioxide won't come out of solution. If some bubbles form in the gel in the last few minutes of the run, they will probably not cause visible distortion. If they form earlier in the run, they may cause distortion because they act as insulators and change the gel temperature. These bubbles form between the gel surface and the cassette.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
All lots of IEF gels that we manufacture exhibit these lines because carrier ampholytes do focus into tight bands extending across the gel and do stain a little. The intensity of these lines varies depending on the ampholytes used. One or more species may be overabundant, leading to more intense ampholyte bands. Usually these are still quite faint compared to the sample.
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For IEF gels, we recommend using an acetic acid transfer buffer. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer:
After the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 minutes. Chill the 0.7% acetic acid that will be used as the transfer buffer. Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (-). This is opposite from the typical western blot with SDS, where the negatively charged protein will migrate toward the anode (+) side during the transfer. Transfer for one hour at 10 volts.
Tip for handling the IEF gel: The 5% polyacrylamide IEF gels tend to be sticky. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position.
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No. An acetic acid fix is not strong enough. However, a 12% trichloroacetic acid (TCA) fix can be substituted.
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Store the fixed, stained, and destained gel strip in 8% acetic acid at 4-25 degrees C in a sealed dish. Do not freeze as this will cause the gel strip to crack.
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No, the SDS will render the protein complex a strong negative charge and therefore an unreliable separation based on isoelectric point. If you have a protein that is moderately soluble, then you can try to solubilize the protein in a non-ionic detergent (between 0.1-0.5%) such as Triton X-100, NP-40, or Tween 20. The non-ionic detergent may help to solubilize the protein, however, this needs to be tested empirically, because the characteristics of the protein such as its purity, concentration, and ionic strength will influence how well it will run on the gel. Very hydrophobic proteins, like membrane proteins, will not work well, even if a non-ionic detergent is added to the sample buffer.
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The cathode buffer goes in the inner (upper) buffer chamber and the anode buffer goes in the outer (lower) buffer chamber of the XCell SureLock Mini Cell.
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It is possible run a denaturing IEF gel, but our Invitrogen pre-cast vertical IEF gels were not designed for denaturing applications. You can try adding ~7 M urea to the sample prior to running to help solubility. This may work for some proteins but not for others. Further, the protein may re-precipitate since there is no urea in the gel itself, resulting in fuzzy bands and smearing. Use of urea in IEF gels or in running buffers is not generally common because the urea does not remain dispersed throughout the gel. It will migrate to a certain point in the gel and then stop moving.
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Yes, you can reduce the protein with DTT or beta-mercaptoethanol prior to running it on an IEF gel, keeping in mind that if the reducing unfolds or opens masked charges, you may see a change in pI.
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We do not recommend adding bromophenol blue to the IEF sample buffer because it is an amphipathic dye that can stick to proteins under native conditions, and cause migration/focusing problems and an increase in current
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0.002% methyl red can be added. It runs at pI=3.6, so it is also good for checking how well the gel is running.
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If the downstream application requires that you don't use amino acids, you can use 100 mM sodium hydroxide. This will create enough ionic strength for isoelectric focusing.
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The use of chilled running buffers will prevent isolated heating and possible gel fissuring at the left and right edges of the gel, thus producing optimal results. The electrophoresis itself can be performed at room temperature.
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The initial voltage step helps to set up the ampholytes in a pH gradient, the second step actually drives the proteins to their pI and the third step is believed to "fine focus" or sharpen the protein bands.
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During IEF, proteins migrate in an electric field until a stable pH gradient is formed and the proteins settle into their pI. A high finishing voltage is applied to focus the proteins into narrow zones. High voltage cannot be used during the initial stages of IEF as movement of carrier ampholytes generates excessive heat. To obtain the best results, IEF is typically performed by gradually increasing the voltage, then maintaining the final focusing voltage for 30 minutes. So, we recommend: 100 volts for the first hour, 200 volts for the second hour, and 500 volts for the last half hour. Since the voltage will increase over time if the run is set at constant current, we recommend setting the current at 5 mA per gel and setting a voltage limit of 500 volts. Alternatively, IEF can be performed at constant power, so the voltage will increase as the current decreases.
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IEF gels have a shelf life of 8 weeks when stored at 4 degrees C.
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The IEF buffers should be stored at 4 degrees C.
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We recommend storing them at 4 degrees C.
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The recommended sample loading volumes and protein loading amounts for the different well formats can be found at:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/recommended-well-loading-volumes-sample-loads.html.
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Our IEF gels do not contain SDS and were not designed for denaturing applications.
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The ratio of acrylamide:bisacrylamide in our IEF gels is 37.5:1.
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Our IEF gels contain 5% acrylamide, 2.6% crosslinker, and 2% ampholytes.
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Invitrogen pH 3-10 IEF gels have a pI performance range of 3.5-8.5 and Invitrogen pH 3-7 IEF gels have a pI performance range of 3.5-6.5.
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*Invitrogen Pre-Cast Vertical IEF gels are excellent for native applications using soluble proteins.
*They can be used for pI determination and confirmation of isoforms of purified products.
*They can readily detect minor changes in a protein due to deamination, phosphorylation or glycosylation, and can resolve different proteins of similar size, which cannot be resolved on standard SDS-PAGE gels. Pre-focusing is not required.
*For a quick 2D analysis, proteins can be separated by pI in the first dimension, using IEF gels, and then by mass in the second dimension, using NuPAGE Bis-Tris gels or Invitrogen Tris-Glycine gels with a 2D-well or by using ZOOM gels for 2D SDS-PAGE.
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Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins on the basis of their isoelectric point (pI). The pI is the pH at which a protein has no net charge and does not move in an electric field. IEF gels effectively create a pH gradient so proteins separate according to their unique pI.
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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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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.
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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)
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.