FAQ
How do I apply the IPG strip to a vertical 2-D gel?
Dip the IPG strip in the SDS electrophoresis buffer to lubricate it. Holding one end of the IPG strip with forceps, carefully draw it across the gel plate until the strip is completely on the glass plate and centered. Using a thin plastic spatula, ruler or spacer, push against the plastic backing of the IPG strip -- not the gel itself -- and slide the strip between the two glass plates and down into contact with the surface of the slab gel. Avoid trapping air bubbles between strip and the slab gel, or between the gel backing and the glass plate and avoid piercing the 2-D gel with the strip. By convention, the acidic (+) or pointed end of the IPG strip is on the left. Seal the strip in place with an agarose sealing solution.
Note: IPG strips pH 6-11 are labeled with a (-) sign at the cathodic side; the cathode is therefore at the barcode end where the print is. All other strips are labeled with a (+) sign at the anodic end.
What causes irregular dye fronts on Multiphor II second-dimension SDS-PAGE gels?
If the buffer strips or the ExcelGel are old this cause irregular dye front. Ensure that the expiration dates on the buffer strips and ExcelGel have not elapsed.
How do I know if isoelectric focusing is proceeding in the IPG strip?
As isoelectric focusing proceeds, the bromophenol blue tracking dye migrates toward the anode. Note that the dye front leaves the IPG strip well before focusing is complete, so clearing of the dye is no indication that the sample is focused. If the dye does not migrate, no current is flowing through the IPG strip. Check all connections and the efficiency of rehydration of the IPG strip.
Should I run my IEF gel under native or denaturing conditions?
IEF can be run in either a native or a denaturing mode. Native IEF is the more convenient option, as precast native IEF gels are available in a variety of pH gradient ranges. This method is also preferred when native protein is required, as when activity staining is to be employed. The use of native IEF, however, is often limited by the fact that many proteins are not soluble at low ionic strength or have low solubility close to their isoelectric point. In these cases, denaturing IEF is employed.
How do I determine the optimum point of sample application on an IPG strip when loading via a sample cup?
The optimal application point depends on the characteristics of the sample. When the proteins of interest have acidic pIs or when SDS has been used in the sample preparation, sample application near the cathode is recommended. Anodic sample application is required with pH 6-11 gradients and preferred when pH 3-10 gradients are used. The optimal application point can vary with the nature of the sample. Empirical determination of the optimal application point is best.
How much salt concentration can be tolerated in an IPG strip sample?
If the sample is rehydrated into the IPG strip, the salt concentration in the final rehydration solution should be lower than 10 mM. "Salt" also includes buffers, charged detergents (e.g. SDS) and charged contaminants (from the sample, sample preparation or breakdown products of additives such as urea). If the sample is applied to IPG strips via sample cups, salt concentrations of up to 50 mM in the sample may be tolerated. However, please note that proteins may precipitate at the sample application point as they abruptly move into a lower-salt environment.
How long should I rehydrate my IPG strips?
A minimum of 10 hours is required for rehydration; overnight is recommended.
Can I store the IPG strips following IEF?
Yes, the IPG strips can be stored at -40 to -80 °C in screw-cap tubes after IEF but prior to equilibration step. The 7 cm strips fit in disposable, 15 ml conical tubes. The 11, 13 and 18 cm strips fit in 25 x 200 mm screw-cap culture tubes. Equilibration Tubes can be used to store 24 cm strips.
How to estimate pI in IPG strips?
Focusing position along the strip gives you a pI search window for database matching.
Do I need to perform a second equilibration step for the IPG strip?
This step is optional, but recommended, when SDS-PAGE is performed in a vertical second-dimension system. It is required when SDS-PAGE is performed on a flatbed second-dimension system, especially when the separation is to be visualized by silver staining.
Iodoacetamide is introduced in a second equilibration step. Iodoacetamide alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis. Protein reoxidation during electrophoresis can result in streaking and other artifacts. Equilibration with iodoacetamide is also used to minimize unwanted reactions of cysteine residues.
What are the dimensions of rehydrated IPG strips?
Rehydrated IPG strips are 3 mm wide and approximately 0.5 mm thick.
What are the possible effects of air bubbles in the final 2-D map?
Air bubbles trapped between the IPG Strip and the second-dimension gel surface may cause vertical gaps in the 2-D map as well as irregular dye fronts. Ensure that no bubbles are trapped between the IPG strip and the top surface of the second-dimension gel. If using a flatbed gel format (i.e. Multiphor II), stroke the plastic backing of the IPG strip gently with a pair of forceps to remove any bubbles.
What is the purpose of the barcodes on the IPG strips?
In high throughput labs the problem of managing all that information coming from hundreds of gels and IPG strips becomes large. A solution to this is collecting all the data in a laboratory information management system (such as our Ettan LWS Software; LWS = Laboratory Workflow System). Scanning barcodes simplies data entry on the IPG strip's lot#, serial#, pH range and length.
What is the recommended current limit per IPG strip?
The recommended current limit is 50 mA per strip.
Which is the anodic end for the IPG strips 6-11?
IPG strips pH 6-11 are labeled with a (-) sign at the cathodic side; the cathode is therefore at the barcode end where the print is. The anodic side is not labeled. All other strips are labeled with a (+) sign at the anodic end.
Why are my IPG strips turning yellow?
Bromophenol blue is a pH indicator dye. It is purple at pH 4.6 (and higher), turning to yellow at pH 3.0. Thus, on a pH 3-10 IPG strip, this dye will turn yellow as it approaches the pH 3 anode.
Why should I remove salts from my IEF sample?
You should avoid having salts, residual buffers and other charged small molecules that carry over from the sample preparation.
Salts disturb the electrophoresis process and must be removed or maintained at as low a concentration as possible.
Salts in the IPG strip result in high conductivity and focusing of the proteins will not occur until the ions have moved to the ends of the strips, prolonging the time required for IEF. Water movement can also result, causing one end of the strip to dry out and the other to swell. In extreme cases, the center of the strip will dry out, leading to sparking and burning there. Also, the salts that eventually pool at the ends of the IPG strip carry all the current in that region, so that the sample proteins do not focus (seen as horizontal streaking in the final result).
If the sample is rehydrated into the IPG strip, the salt concentration in the rehydration solution should be lower than 10 mM.
If the sample is applied in sample cups, salt concentrations of up to 50 mM in the sample may be tolerated. However, proteins may precipitate at the sample application point as they abruptly move into a lower-salt environment.
Would a sample that is poorly soluble in rehydration solution cause my gel to have horizontal streaking?
Yes. Proteins that are not soluble cannot migrate, and therefore cannot focus. Increase the concentration of the solubilizing components in the rehydration solution.
- Commonly 8 M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary.
- CHAPS, Triton X-100, or NP-40 are the neutral detergents most commonly used.
Use at a total combined concentration not exceeding 4% (v/v).
- DTT or DTE (20 to 100 mM) is the reducing agent most commonly used.
- The recommended IPG Buffer (carrier ampholyte mixture) concentration for the IPGphor system is 0.5%,
but up to 2% can be added if sample solubilization remains a problem.
What internal standards can I use in 2-D electrophoresis?
Experienced researchers often find that the endogenous, well-characterized, abundant proteins in their samples serve as excellent internal standards as opposed to adding contaminating proteins to their 2-D samples. If molecular weight standards are desired, these can be run adjacent to the IPG strip on the 2-D gel.
Which ExcelGel buffer strip is the anode one?
The orange one is the anode buffer strip.
Can I run IPG strips under native conditions?
That is entirely dependent upon the nature of your sample. Keep in mind that IEF is a high voltage application, and therefore, samples must be desalted to avoid generating high current and high heat. Most proteins become insoluble at low salt, and therefore, uncharged denaturants such as 8M urea and 1-4% nonionic detergents must be introduced to maintain solubility of the proteins. If these uncharged denaturants are not required to maintain sample solubility, then you can run the IPG strips under "native conditions."
Note: A subsequent observation of streaking in the horizontal dimension is an indication that the sample is insoluble. If the sample has not precipitated, pelleted or become turbid is NOT sufficient indication that the sample is still soluble.
How do I choose the pH range of an IPG strip?
A pH 3-10L or 3-10NL IPG strip will display the widest range of proteins on a single 2-D gel and is a good starting point for unknown samples. The pH 4-7 and pH 6-11 IPG strips offer improved resolution in the acidic and basic ends of the spectrum, respectively. The narrowest pH ranges (1 pH unit wide) are used for higher resolution separations in a particular pH range.
See more in Selection guide for Immobiline DryStrip gels
How do I choose the appropriate length of IPG strips?
Choose shorter strips for fast, cost-effective screening purposes (for a quick overview) or when only the most abundant proteins are of interest (as in pre-fractionated protein complexes). The shortest IPG strips give the fastest results, but the sample load is limited. Use longer strips for maximal resolution and loading capacity. Longer strips allow the detection of more spots and facilitate the selection and identification of proteins in the spots. But remember that both longer focusing times in the first dimension and longer separation run times in the second dimension will be required since long IPG strips are normally loaded onto larger 2-D gels.
How can I apply my sample into the IPG strip?
Sample can be applied either by including it in the rehydration solution (rehydration loading) or by applying it directly to the rehydrated Immobiline DryStrip via sample cups or a paper bridge.
1. Rehydration loading.
Rehydration loading offers such advantages as loading and separation of large sample volumes (>100 µl), larger sample amounts, and more dilute samples. Because there is no discrete application point, this method eliminates the formation of precipitates at the application point that may occur when loading using sample cups. Also, the method is technically simpler than the others, avoiding problems of leakage that can occur when using sample cups. The total rehydration volume is dependent on the strip length (125 µl for 7 cm strips, 200 µl for 11 cm strips, 250 µl for 13 cm strips, 340 µl for 18 cm strips and 450 µl for 24 cm strips).
If the Manifold is used, Immobiline DryStrip gels must be rehydrated prior to IEF using IPGbox and the Reswell Tray. Rehydration can take place with or without the sample included.
2. Cup loading with manifold.
There are cases when it may be preferable to load the sample following rehydration, immediately prior to IEF. For example, if proteolysis or other protein modifications are a concern, overnight rehydration with sample may not be desirable. The Manifold provides a convenient means to load samples under such circumstances. Cup loading using the Manifold is recommended for sample volumes up to 150 µl, and a maximum protein concentration of 150 µg protein/150 µl sample solution (150 µl is the volume of the cup). Larger sample loads can lead to increased protein precipitation at the point of application. Anodic cup loading has been found to improve protein 2-D spot patterns with basic Immobiline DryStrip gels (pH 6-9, pH 6-11, and pH 7-11 NL). Under conditions where substantial water transport (electroendosmosis) accompanies focusing, such as with protein loads in excess of 1 mg, the face-up mode frequently yields better resolution. See section 2.3.4 of our 2-D Electrophoresis Handbook for more information on face-up mode.
3. Paper-bridge loading.
Paper-bridge loading is ideal for very large sample volumes and preparative electrophoresis, and is particularly applicable when using basic pH intervals (pH 6-9, pH 6-11, and pH 7-11 NL). Paper-bridge loading can also be performed in the Manifold. Using 18- or 24-cm Immobiline DryStrip gels, up to 450 µl can be applied using the paper-bridge method.
Note: Alternatively, sample may be added to rehydrated strips by pipetting sample into either or both of the lateral wells at either end of the strip holder. Up to 7.5 µl of sample solution can be added to each side (i.e. 15 µl per well or 30 µl total if both sides of both wells are used). Introduce the sample below the IPG cover fluid. The IPG strip gel backing is impermeable; do not apply the sample to the back of the strip.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Select symptom:
Possible cause | Suggested remedy |
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Low current at start of run |
IPG strip not properly rehydrated |
Low current because the EPS 3501 XL Power Supply cannot detect the low μA low range current and shuts off. |
Because the EPS 3501 XL Power Supply can operate under very low currents, it is recommended for use with Immobiline DryStrip Kit and Immobiline DryStrip gels. Make sure the low-current shutoff has been bypassed (see 3501 XL Power Supply instructions). IPG runs may start in a current range that is not detectable by the EPS 3501 XL Power Supply. |
Low current due to IPG Buffer omitted from rehydration solution. |
Always include IPG Buffer or Pharmalyte in the rehydration solution |
No current at start of run |
Wrong sample cups used leading to the sample cup slicing into the gel, blocking current. Do not use the strip holder sample cups on the manifold. |
No current later in the run |
IPG strips have dried out during the course of the run. Allow the strips at least 10 hours to rehydrate prior to running. Remember to cover the rehydrated strips with DryStrip cover fluid prior to running. Also, electroendosmosis due to excessive amounts of charged material in the sample or additives may be drying out the center of the strip. Desalt your samples and deionize your urea, thiourea and other additives prior to use. |
Possible cause | Suggested remedy |
---|---|
Conductivity of the sample/IPG strips is too high |
Ensure that the sample is adequately desalted and that additives are neutral (and haven't degraded). Salts pool at the ends of the strips, and then water migrates towards the ends of the strips, too, in order to try to dilute out the salts. The center of the strips dries out and arcing can occur across this gap. |
IPG strip dried out |
IPG strip dried out because the strip was not submerged in DryStrip Cover Fluid. |
Caused by strong electroendosmosis. During SDS equilibration, the Immobiline gel becomes strongly negatively charged due to the fixed buffer groups. After the transfer of water in the direction of the cathode, the Immobiline and SDS gel dry out along the contact line at the anode edge of the gel strip. |
Equilibrate twice, for 15 minutes each time, while shaking in 1-2% SDS, 0.15 M Tris-HCl (pH 8.8), 6 M urea, 30% glycerin, 0.1% DTT, bromophenol blue. Modified equilibration buffer compensates for electroendosmosis effect in around 1 hour (during this time all proteins have left the IPG strip). After 1 hour, take IPG strips off and shift the SDS buffer strip so that it overlaps the gel over the contact line. |
Possible cause | Suggested remedy |
---|---|
High current and therefore high heat at the ends of the strips, due to charged material in the IPG strip |
1. The samples must be cleaned up. See the 2-D Principles & Methods Guide. Most 2-D researchers use a TCA/acetone precipitation protocol to remove salts and buffers and most ionic contaminants. |
Possible cause | Suggested remedy |
---|---|
Too much charged contaminants in the sample. Charged contaminants accumulate in high concentrations near the electrodes and consume the current. Proteins and the dye will therefore migrate very slowly. |
1. Remove contaminants, refer to 2-D Principles & Methods Guide |
Possible cause | Suggested remedy |
---|---|
Excess charged contaminants in the sample and/or the rehydration solution |
1. Modify sample preparation to limit contaminants, refer to 2-D Principles & Methods Guide |
Possible cause | Suggested remedy |
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Incomplete rehydration |
Rehydrate for the recommended time (10 hours or more) Rehydrate under cover fluid. Ensure complete contact of the rehydration solution along the entire length of the IPG strip. |
Too much charged impurities in the sample |
Clean up the sample using the 2-D Clean-Up Kit. |
Unequal contact between electrode and paper electrode strip, or paper electrode strip and IPG strips. |
Ensure that the paper electrode strip is resting completely on gel, and not just on the GelBond backing of the IPG strip. |
Wire is not clean and taut |
Inspect the electrode wires for signs of wear (even Pt wires require replacement eventually) or surface carbon deposits (left behind by burnt gels in previous runs). The wire should be clean and taut. |
- |
Extend the time for focusing. The window for good focusing is generally quite large; focusing during longer time enable the slower focusing strip focuses while the faster focusing strip doesn't over-focus. |
Possible cause | Suggested remedy |
---|---|
Depending on the Immobiline DryStrip gel pH interval and the pH of the rehydration solution, either the basic end or the acidic end will swell faster than the other. The strip may not necessarily be of an even thickness following rehydration. |
At the start of rehydration, ensure that the rehydration of strips is evenly distributed under the Immobiline DryStrip gel. Move the gel strip back and forth to aid distribution. The gel strip should float on the rehydration solution. |
Unopened Immobiline DryStrip gel package was stored at or above room temperature for too long. |
Store Immobiline DryStrip gels sealed at a temperature below -20 °C. |
Incorrect volume of rehydration solution used. |
Make sure the correct amount of rehydration solution is added to the channel in the Immobiline DryStrip Reswelling Tray. |
Rehydration time is too short. |
Rehydrate the Immobiline DryStrip gels for at least 10 h. |
Possible cause | Suggested remedy |
---|---|
Aggregates and complexes in the sample |
The addition of a maximum of 0.8% (w/v) carrier ampholytes (IPG buffer) to the sample increases solubility without increasing the formation of artifacts in the Immobiline gel. |
Protein and/or salt concentration in the sample too high |
Dilute the sample. It is preferable to use a larger sample volume than a concentrated solution. Keep the contact area between sample solution and gel surface as small as possible (i.e., use sample cups). |
Initial field strength too high |
Do not prefocus (pH gradient already exists). When carrying out IEF on individual bands, use the voltage to control the field strength. Use maximum 40 V/cm for 1 hour and then increase voltage. |
Possible cause | Suggested remedy |
---|---|
Temperature too low during IEF |
Focus at 15 °C |
Surface is drying out |
Ensure that the strips are covered with DryStrip Cover Fluid. Add 0.5% Triton X100 or Nonidet NP-40 to the rehydration solution. |
Charged contaminants in sample |
Clean the sample by use of 2-D Clean-Up kit or by TCA/acetone precipitation. |
Urea has degraded during storage |
Urea can easily break down during storage. Check that the urea crystals have not picked up water from the air. If the urea crystals are damp, de-ionize the urea with Amberlite before use, or use a new bottle of urea |