# Isoelectric Focusing (IEF) for Protein Analysis ## Quick Answer - Separate proteins by isoelectric point using an immobilized pH gradient strip, then focus at high voltage until current stabilizes to indicate completion. - Prevent protein precipitation at the pI by adding urea, thiourea, CHAPS, and dithiothreitol to the rehydration buffer and keeping samples at low concentration. - Resolution limits depend on gradient width, strip length, and sample complexity, so choose narrow gradients for closely spaced protein variants and expect some protein loss at the pI. ## At a Glance | Parameter | Typical Choice | Effect on Resolution | Practical Note | | --- | --- | --- | --- | | pH gradient | 3 to 10 for survey, 4 to 7 or narrower for detail | Narrow gradients spread proteins across more millimeters per pH unit | Use narrow gradients when target proteins focus in a known pH window | | Strip length | 7 cm for screening, 18 to 24 cm for high resolution | Longer strips increase separation distance and peak capacity | Longer strips require longer focusing times and higher voltages | | Sample load | 50 to 100 micrograms for silver stain, 200 to 500 micrograms for Coomassie | Overloading causes horizontal streaking and distorted bands | Start low and increase only if detection sensitivity requires it | | Focusing time | 20 to 60 kilovolt hours depending on strip length | Insufficient time gives blurred bands, excess time causes water migration | Monitor current decay and stop when the current reaches a stable low value | ## Understanding Isoelectric Focusing Isoelectric focusing separates proteins according to their isoelectric point, the pH value at which a protein carries no net electric charge. At a pH below its p, a protein carries a net positive charge and migrates toward the cathode. At a pH above its p, the protein carries a net negative charge and migrates toward the anode. When a protein reaches the region of the gel where the pH equals its p, its net charge becomes zero and it stops migrating. This concentrating effect makes IEF a high-resolution separation method for protein variants that differ by a single charge. The separation medium contains ampholytes, which are mixtures of small molecules with closely spaced p values. When an electric field is applied, the ampholytes arrange themselves to form a continuous pH gradient. Proteins migrate through this gradient until they reach their p position. The pH gradient can be created in two ways. Carrier ampholyte gels generate the gradient during the focusing run as the ampholytes establish their positions. Immobilized pH gradient strips have the pH gradient covalently attached to the gel matrix, which provides a more stable gradient that does not drift during extended focusing. The choice between carrier ampholyte and immobilized pH gradient systems affects reproducibility and resolution. Immobilized pH gradients resist the cathodic drift that can occur in carrier ampholyte systems, and they allow higher protein loads without the risk of gradient collapse. Most modern two-dimensional gel electrophoresis workflows use immobilized pH gradient strips for the first dimension. The National Library of Medicine provides access to laboratory-method references that describe the standard molecular-biology workflows used in protein analysis, including the general framework for electrophoretic separations. ## Core Principles of Protein Separation by p ### The Relationship Between pH and Protein Net Charge A protein is a polymer of amino acids, some of which carry ionizable side chains. The side chains of aspartic acid and glutamic acid can lose a proton and become negatively charged. The side chains of lysine, arginine, and histidine can gain a proton and become positively charged. The amino terminus and carboxyl terminus also contribute to the overall charge. The net charge of the protein depends on the pH of the surrounding solution because each ionizable group has its own pKa value. At a pH equal to the p of the protein, the number of positive charges equals the number of negative charges. The protein has no net charge and does not migrate in an electric field. At any other pH, the protein carries a net charge and will migrate toward the electrode of opposite polarity. The steepness of the charge change around the p depends on the number of ionizable groups and their pKa values. A protein with many histidine residues, which have a pKa near neutral pH, will have a sharp charge transition near its p. A protein with few ionizable groups will have a shallow charge transition. ### The Role of Ampholytes in Gradient Formation Ampholytes are the molecules that create the pH gradient in carrier ampholyte systems. These molecules are mixtures of polyamino-polycarboxylic acids with a range of p values. When the electric field is applied, each ampholyte species migrates until it reaches a position where the local pH equals its own p. The ampholytes then carry no net charge and stop migrating. The result is a series of ampholyte molecules arranged in order of their p values, creating a smooth pH gradient across the gel. The quality of the gradient depends on the ampholyte mixture. A good ampholyte mixture contains many species with closely spaced p values so that the gradient is smooth instead of stepwise. The ampholytes also need to have sufficient buffering capacity to maintain the pH at each position. The concentration of ampholytes in the gel affects the shape of the gradient and the speed of focusing. Too little ampholyte gives a shallow gradient with poor buffering. Too much ampholyte can cause the gradient to be distorted or can interfere with protein detection. ### Immobilized pH Gradients and Their Stability Immobilized pH gradient strips use a different mechanism to create the pH gradient. The gel matrix contains acrylamide monomers that have been modified with buffering groups. These groups are covalently attached to the gel during polymerization, so the pH gradient is fixed in the gel. The gradient does not drift during the run because the buffering groups cannot migrate. This stability is the main advantage of immobilized pH gradients over carrier ampholyte systems. The immobilized pH gradient is created by mixing two solutions with different buffering compositions in a gradient mixer during gel casting. The resulting gel has a linear or nonlinear pH gradient across its length. The gradient range is selected based on the expected p values of the proteins of interest. A broad gradient from pH 3 to 10 is useful for a first survey of a complex sample. A narrow gradient such as pH 4 to 7 provides higher resolution for proteins that focus in that range. The practical consequence of the immobilized gradient is that the focusing time can be extended without the risk of gradient collapse. The proteins will reach their p and stop migrating, and the gradient will remain stable. This stability allows the use of higher voltages and longer focusing times to achieve sharper bands. The tradeoff is that the strips are more expensive than carrier ampholyte gels and require a dedicated rehydration and focusing apparatus. ## Practical Workflow for IEF ### Sample Preparation and Solubilization The quality of the IEF result depends on the quality of the sample. Proteins must be fully solubilized and denatured before focusing. The standard solubilization buffer contains urea, thiourea, a detergent such as CHAPS, and a reducing agent such as dithiothreitol. Urea at high concentration disrupts hydrogen bonds and denatures proteins. Thiourea improves the solubility of hydrophobic proteins. CHAPS is a zwitterionic detergent that keeps denatured proteins in solution. Dithiothreitol reduces disulfide bonds and prevents protein aggregation. The sample buffer should be prepared fresh and used within a short time. Urea in solution slowly decomposes to form isocyanate, which can carbamylate protein amino groups and alter their p values. This modification creates charge trains that appear as a series of spots with slightly different p values on a two-dimensional gel. To minimize this artifact, prepare the urea buffer immediately before use and do not heat the sample above 30 degrees Celsius. The protein concentration of the sample should be measured before loading. The amount of protein that can be loaded onto an immobilized pH gradient strip depends on the strip length and the pH range. A 7 centimeter strip can typically handle 50 to 100 micrograms of protein. An 18 centimeter strip can handle 200 to 500 micrograms. Overloading the strip causes the proteins to precipitate at their p and creates horizontal streaks. Underloading gives faint spots that are difficult to detect. ### Rehydration and Loading The immobilized pH gradient strip must be rehydrated before the sample is applied. The rehydration solution contains urea, thiourea, CHAPS, dithiothreitol, ampholytes, and a trace of bromophenol blue as a tracking dye. The sample is mixed with the rehydration solution and applied to the strip. The strip is then placed in a rehydration tray and covered with mineral oil to prevent evaporation. The rehydration time depends on the strip length and the gel thickness. A typical rehydration time is 12 to 16 hours at room temperature. The rehydration can be done with the sample included in the rehydration solution, which is called in-gel rehydration. Alternatively, the sample can be applied to the strip after rehydration using a sample cup. In-gel rehydration is simpler and gives more reproducible results for most samples. The volume of the rehydration solution must be matched to the strip size. A 7 centimeter strip requires approximately 125 microliters. An 18 centimeter strip requires approximately 340 microliters. The strip should absorb the entire volume during rehydration. If the strip does not absorb all the solution, the excess can be removed with a pipette. The mineral oil overlay prevents the strip from drying out during the run. ### Focusing Conditions The focusing run is performed in a dedicated IEF apparatus that can apply a programmed voltage profile. The voltage is increased gradually to prevent the proteins from moving too quickly and precipitating. A typical program starts at a low voltage such as 200 volts for 1 hour, then increases to 500 volts for 1 hour, then to 1000 volts for 1 hour, and finally to 8000 volts for the remaining time. The total focusing time is expressed in kilovolt-hours, which is the product of the voltage and the time. The focusing time depends on the strip length and the pH range. A 7 centimeter strip with a broad pH range may require 20 to 30 kilovolt-hours. An 18 centimeter strip with a narrow pH range may require 50 to 80 kilovolt-hours. The current should decrease during the run as the proteins reach their p and stop migrating. The run is complete when the current reaches a low stable value and does not decrease further. The temperature of the focusing tray should be maintained at 20 degrees Celsius. Higher temperatures can cause the urea to decompose and can lead to protein precipitation. Lower temperatures can cause the urea to crystallize. The mineral oil overlay prevents the strip from drying out and provides thermal contact with the cooling plate. ### Equilibration and Transfer to Second Dimension After focusing, the immobilized pH gradient strip must be equilibrated before it can be applied to a second-dimension gel. The equilibration step replaces the focusing buffer with a buffer that is compatible with the second-dimension SDS-PAGE. The equilibration buffer contains SDS, glycerol, Tris buffer, and dithiothreitol. The SDS binds to the proteins and gives them a uniform negative charge per unit mass. The equilibration is done in two steps. The first step uses a buffer containing dithiothreitol to reduce disulfide bonds. The second step uses a buffer containing iodoacetamide to alkylate the reduced cysteine residues. The alkylation prevents the disulfide bonds from re-forming during the second-dimension run. Each equilibration step takes 15 minutes at room temperature with gentle agitation. The equilibrated strip is then placed on top of the second-dimension gel and sealed with agarose. The proteins are separated by molecular weight in the second dimension. The result is a two-dimensional gel with proteins separated by p in the first dimension and by molecular weight in the second dimension. ## Options and Tradeoffs in IEF ### Carrier Ampholyte versus Immobilized pH Gradient The choice between carrier ampholyte and immobilized pH gradient systems affects the resolution, the reproducibility, and the practical complexity of the experiment. Carrier ampholyte gels are simpler to prepare and do not require a dedicated rehydration step. They are useful for analytical separations where the pH gradient does not need to be exactly reproducible. The gradient is formed during the run and can be affected by the sample composition and the focusing time. Immobilized pH gradient strips provide a more stable and reproducible gradient. The gradient is fixed in the gel, so the pH at each position is known and consistent between runs. This reproducibility is important for comparing samples across experiments. The strips are available in a wide range of pH gradients, from broad ranges such as pH 3 to 10 to narrow ranges such as pH 4 to 5. The narrow ranges provide higher resolution for proteins with similar p values. The trade-off is that immobilized pH gradient strips are more expensive and require a dedicated rehydration and focusing apparatus. The strips also have a limited sample capacity. The user must choose the strip length and pH range based on the number of proteins in the sample and the resolution needed. ### Narrow versus Broad pH Gradients The pH gradient range determines the resolution of the separation. A broad gradient such as pH 3 to 10 spreads the proteins across a wide pH range, so each protein occupies a small fraction of the gel length. This gradient is useful for a first survey of a complex sample to see the overall distribution of proteins. The resolution is lower because the distance between two proteins with similar p values is small. A narrow gradient such as pH 4 to 7 spreads the proteins across a smaller pH range. The distance between two proteins with similar p values is larger, so the resolution is higher. This gradient is useful for separating protein variants that differ by a single charge. The tradeoff is that proteins with p values outside the gradient range will not focus and will be lost. The choice of gradient range should be based on the known p values of the target proteins. If the p values are not known, a broad gradient is a reasonable starting point. The user can then choose a narrow gradient that covers the p range of interest for a second run. ### Sample Load and Detection Sensitivity The amount of protein loaded onto the strip affects the detection sensitivity and the resolution. A higher load gives more protein per spot, which makes detection easier. However, a higher load can also cause the proteins to precipitate at their p, especially if the protein is hydrophobic or has a high molecular weight. The precipitation creates horizontal streaks that obscure the spots. The detection method also affects the optimal load. Coomassie staining requires a higher protein load than silver staining. A typical Coomassie-stained gel requires 200 to 500 micrograms of protein. A silver-stained gel can detect 50 to 100 micrograms. The user should choose the load based on the detection method and the expected abundance of the target protein. The load can be increased by using a longer strip. An 18 centimeter strip has a higher capacity than a 7 centimeter strip. The longer strip also provides a higher resolution because the proteins are spread over a longer distance. The tradeoff is that the longer strip requires a longer focusing time and a more expensive apparatus. ## Observations and Measurements ### Monitoring the Focusing Run The progress of the focusing run can be monitored by observing the current and the voltage. The current should decrease as the proteins reach their p and stop migrating. The current is initially high because the ampholytes and the proteins are moving. As the run progresses, the current decreases and eventually reaches a low stable value. The run is complete when the current does not change over a period of time. The tracking dye, usually bromophenol blue, migrates to the anode during the run. The dye is a small molecule that does not focus at a specific p. It moves to the anode and can be used to monitor the progress of the run. The dye should reach the anode end of the strip by the end of the run. If the dye has not reached the anode, the focusing time may be insufficient. The voltage profile can be adjusted to improve the focusing. A slow voltage ramp prevents the proteins from moving too quickly and generating heat. A fast ramp can cause the proteins to precipitate. The user can also increase the final voltage to sharpen the bands. The final voltage is limited by the capacity of the power supply and the heat generated by the gel. ### Recording the Results The results of the IEF run are recorded as the position of the protein bands on the strip. The p of each protein can be estimated by comparing the position of the band to the position of the p markers. The p markers are a mixture of proteins with known p values that are run alongside the sample. The markers are detected by staining or by a specific detection method. The position of the band is measured from the anode end of the strip. The p of the protein is then read from the calibration curve of the p markers. The calibration curve is a plot of the p of the markers against their position on the strip. The curve is linear for a well-formed gradient. The reproducibility of the p measurement depends on the stability of the gradient and the accuracy of the position measurement. The immobilized pH gradient strips provide a more reproducible gradient than carrier ampholyte gels. The position of the band can be measured with a gel scanner or by visual inspection. The measurement error is typically 0.1 to 0.2 pH units. ### Quality Controls The quality of the IEF run can be assessed by the appearance of the bands. A well-focused protein appears as a sharp, narrow band. A poorly focused protein appears as a broad band or a streak. The presence of horizontal streaks indicates that the protein precipitated at the p. The presence of vertical streaks indicates that the protein did not focus completely. The reproducibility of the run can be assessed by running the same sample on two different strips. The p values of the proteins should be the same within the measurement error. The band intensity should also be similar. The reproducibility is affected by the sample preparation, the rehydration, and the focusing conditions. The quality of the sample can be assessed by the presence of the protein. The protein should be present in the sample at a concentration that is sufficient for detection. The sample should be free of contaminants that can interfere with the focusing. The contaminants can include salts, detergents, and nucleic acids. ## Common Failure Patterns and Their Causes ### Protein Precipitation at the p The most common failure in IEF is protein precipitation at the p. When a protein reaches its p, it has no net charge and is less soluble in the aqueous buffer. The protein can aggregate and precipitate, forming a horizontal streak on the gel. The precipitation is more likely for proteins that are hydrophobic or have a high molecular weight. The precipitation can be reduced by adding a higher concentration of urea and thiourea to the rehydration buffer. The urea and thiourea denature the protein and keep it in solution. The detergent CHAPS also helps to keep the protein soluble. The reducing agent dithiothreitol prevents the formation of disulfide bonds that can cause aggregation. The precipitation can also be reduced by lowering the protein load. A lower load reduces the concentration of the protein at the p and reduces the chance of precipitation. The user can also use a longer strip to spread the protein over a larger area. ### Uneven pH Gradient The pH gradient can be uneven if the ampholytes are not properly mixed or if the gradient is not stable. The uneven gradient causes the proteins to focus at the wrong position or to not focus at all. The gradient can be uneven in carrier ampholyte systems if the ampholytes are not of good quality or if the focusing time is too short. The gradient can also be uneven in immobilized pH gradient strips if the strip is not rehydrated properly. The rehydration should be uniform across the strip. The strip should be placed in the rehydration tray with the gel side down and the plastic backing up. The rehydration solution should be applied evenly. The gradient can be affected by the temperature. The pH of the buffer changes with temperature. The focusing should be done at a constant temperature of 20 degrees Celsius. The temperature should be maintained by the cooling plate of the apparatus. ### Poor Resolution The poor resolution can be caused by a number of factors. The pH gradient may be too broad for the proteins of interest. The proteins with similar p values will not be separated if the gradient is too broad. The user can choose a narrow gradient to improve the resolution. The poor resolution can also be caused by the focusing time. If the focusing time is too short, the proteins will not reach their p and will appear as broad bands. If the focusing time is too long, the proteins can diffuse and the bands can broaden. The optimal focusing time is determined by the kilovolt-hour. The poor resolution can be caused by the sample load. A high load can cause the proteins to precipitate and to form streaks. The streaks can obscure the bands and reduce the resolution. The user can reduce the load to improve the resolution. ### The Cathodic Drift The cathodic drift is a phenomenon that occurs in carrier ampholyte systems. The ampholytes can migrate toward the cathode over time, which causes the pH gradient to shift. The shift can cause the proteins to move to the wrong positions. The cathodic drift is more pronounced in the basic region of the gradient. The cathodic drift can be reduced by using immobilized pH gradient strips. The gradient is fixed in the gel and does not drift. The immobilized gradient is more stable than the carrier ampholyte gradient. The cathodic drift can also be reduced by using a shorter focusing time. The drift is more pronounced with longer focusing times. The user can use the minimum focusing time that gives a good resolution. ## Quality Controls and Reproducibility ### The Use of p Markers The p markers are a mixture of proteins with known p values. The markers are used to calibrate the pH gradient and to verify the accuracy of the p measurements. The markers are included in the sample or run in a separate lane. The markers should be chosen to cover the pH range of the gradient. The p markers are used to create a calibration curve. The curve is a plot of the p of the markers against their position on the gel. The curve is used to estimate the p of the unknown proteins. The curve should be linear if the gradient is well-formed. The p markers are also used to assess the reproducibility of the run. The position of the markers should be the same in different runs. The markers can be used to correct for the run-to-run variation. ### The Use of Replicates The reproducibility of the IEF can be assessed by running the same sample in multiple replicates. The replicates should be run on the same day and under the same conditions. The position of the bands should be the same in the replicates. The intensity of the bands should also be similar. The replicates are used to assess the technical variation of the method. The technical variation is the variation that is caused by the method itself. The technical variation can be reduced by standardizing the protocol and by using the same equipment and reagents. The replicates are also used to assess the biological variation of the sample. The biological variation is the variation that is caused by the differences between the samples. The biological variation is the variation that is of interest in the experiment. ### The Use of Standards The standards are used to calibrate the IEF system. The standards are a mixture of proteins with known p values. The standards are used to verify that the pH gradient is correct and that the focusing is working properly. The standards are run in a separate lane or in a separate gel. The standards are used to check the performance of the system. The standards should focus at the expected positions. If the standards do not focus at the expected positions, the system is not working properly. The user should check the pH gradient, the focusing time, and the temperature. The standards are also used to compare the results from different runs. The standards provide a reference for the position of the proteins. The standards can be used to correct the differences between the runs. ## Safety and Regulatory Context ### Biosafety Considerations The IEF procedure involves the use of chemicals that can be hazardous. The urea and thiourea are irritants and can be harmful if inhaled or ingested. The CHAPS is a detergent that can cause skin and eye irritation. The dithiothreitol is a reducing agent that can be harmful if inhaled or ingested. The acrylamide is a neurotoxin and a suspected carcinogen. The user should wear appropriate personal protective equipment, including gloves, a lab coat, and safety glasses. The user should work in a well-ventilated area. The user should avoid inhaling the dust from the acrylamide and the other chemicals. The user should wash the hands after handling the chemicals. The user should follow the biosafety guidelines of the institution. The CDC and NIH provide the biosafety principles and the risk assessment for the laboratory work. The user should be aware of the biosafety level of the laboratory and the appropriate containment procedures. ### Electrical Safety The IEF apparatus uses high voltage, which can be dangerous. The voltage can be as high as 8000 volts. The user should be careful when handling the apparatus. The user should not touch the electrodes or the gel while the power is on. The user should turn off the power before handling the gel. The user should use the apparatus according to the manufacturer's instructions. The apparatus should be properly grounded. The user should not use the apparatus if it is damaged. The user should report any damage to the supervisor. The user should be aware of the risk of electric shock. The user should not work alone in the laboratory. The user should be trained in the safe use of the apparatus. ### The Regulatory Context The IEF procedure is a laboratory method that is used for research and diagnostic purposes. The method is not regulated by a specific federal regulation. The laboratory should follow the institutional biosafety and the chemical safety regulations. The NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules apply to the research that involves the recombinant or synthetic nucleic acid molecules. The IEF procedure may be used to analyze the proteins that are produced by the recombinant or synthetic nucleic acid molecules. The user should be aware of the NIH guidelines and the institutional oversight. The NIST provides the measurement science and the standards for the laboratory. The NIST standards can be used to calibrate the equipment and to ensure the accuracy of the measurements. The user should be aware of the NIST standards and the quality control procedures. ## Professional Escalation Criteria The user should escalate the problem to a supervisor or a specialist if the IEF results are not reproducible or if the resolution is poor. The user should also escalate the problem if the protein precipitation is severe and cannot be resolved by the troubleshooting steps. The user should also escalate the problem if the pH gradient is not stable or if the p markers do not focus at the expected positions. The user should also escalate the problem if the IEF is used for a diagnostic or a clinical purpose. The IEF is a research method and is not a validated diagnostic test. The user should not use the IEF for the diagnosis of a disease without the appropriate validation. The user should also escalate the problem if the IEF is used for the quality control of a product. The IEF is a research method and is not a validated quality control method. The user should validate the IEF for the specific product and the specific purpose. The user should also escalate the problem if the IEF is used for the regulatory submission. The IEF is a research method and is not a validated regulatory method. The user should validate the IEF for the specific regulatory purpose. ## Frequently Asked Questions ### What is the difference between isoelectric focusing and SDS-PAGE? Isoelectric focusing separates proteins by their isoelectric point, which is the pH at which the protein has no net charge. SDS-PAGE separates proteins by their molecular weight. In IEF, the protein migrates in a pH gradient until it reaches its p and stops. In SDS-PAGE, the protein is denatured and coated with SDS, which gives it a uniform negative charge, and it migrates through a polyacrylamide gel based on its size. The two methods are often combined in two-dimensional gel electrophoresis, where IEF is the first dimension and SDS-PAGE is the second dimension. ### How do I choose the pH gradient for my IEF experiment? The pH gradient should cover the p range of the proteins of interest. If the p values are not known, a broad gradient such as pH 3 to 10 is a reasonable starting point. If the p values are known, a narrow gradient that covers the p range will provide higher resolution. The narrow gradient spreads the proteins over a larger distance, which makes it easier to separate proteins with similar p values. The gradient range is also limited by the length of the strip and the focusing time. ### Why does my protein precipitate at the p? The protein precipitates at the p because it has no net charge and is less soluble in the aqueous solution. The precipitation is more likely for hydrophobic proteins or proteins with a high molecular weight. The precipitation can be reduced by increasing the concentration of urea and thiourea in the rehydration buffer, by adding CHAPS, and by reducing the protein load. The precipitation can also be reduced by using a longer strip to spread the protein over a larger area. ### How do I know when the focusing is complete? The focusing is complete when the current reaches a low stable value and does not change over a period of time. The current decreases as the proteins reach their p and stop migrating. The tracking dye, bromophenol, should also reach the anode end of the strip. The focusing time is measured in kilovolt-hours, which is the product of the voltage and the time. The focusing time depends on the strip length and the pH range. ### What is the difference between carrier ampholyte and immobilized pH gradient? Carrier ampholyte systems use small molecules that form a pH gradient during the focusing run. The gradient is formed by the ampholytes as they migrate to their p. The gradient is not fixed and can drift during the run. Immobilized pH gradient strips have the pH gradient covalently attached to the gel matrix. The gradient is fixed and does not drift. The immobilized gradient is more stable and provides a more reproducible separation. ### How much protein should I load onto the strip? The amount of protein depends on the strip length and the detection method. A 7 centimeter strip can typically handle 50 to 100 micrograms of protein. An 18 centimeter strip can handle 200 to 500 micrograms. The detection method also affects the load. Coomassie detection requires a higher load than silver detection. The user should start with a low load and increase it only if the detection sensitivity requires it. ### What is the purpose of the equilibration step? The equilibration step is used to prepare the focused strip for the second-dimension SDS-PAGE. The equilibration buffer contains SDS, glycerol, and a reducing agent. The SDS binds to the proteins and gives them a uniform negative charge. The reducing agent breaks the disulfide bonds. The equilibration is done in two steps, first with dithiothreitol and then with iodoacetamide. The iodoacetamide prevents the disulfide bonds from re-forming during the second-dimension run. ### How do I measure the p of a protein from the IEF gel? The p of a protein is measured by comparing its position on the gel to the position of the p markers. The p markers are a mixture of proteins with known p values. The position of the markers is plotted against their p values to create a calibration curve. The p of the unknown protein is then read from the calibration curve at the position of the band. The measurement error is typically 0.1 to 0.2 pH units. ## Using the Evidence | Source | Best use in this topic | Important limitation | |---|---|---| | [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books) | official guidance | Check the linked page for current local requirements | | [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html) | official guidance | Check the linked page for current local requirements | | [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules) | official guidance | Check the linked page for current local requirements | ## Related Molecular Biology Guides - [Protein Quantification: Methods, Principles, and Best Practices](/knowledge/molecular-biology/quantify-proteins) - [Protein Crystallization: Principles, Methods, and Applications](/knowledge/molecular-biology/protein-crystallization) - [Quantification of Protein Concentration: Methods and Principles](/knowledge/molecular-biology/quantification-of-protein-concentration) - [Quantitative Analysis of Proteins: Methods and Applications](/knowledge/molecular-biology/quantitative-analysis-of-proteins) - [Co-Immunoprecipitation Assay: Principles and Protocol](/knowledge/molecular-biology/co-immunoprecipitation-assay) ## References and Further Reading - [Molecular Biology and Laboratory Methods](https://www.ncbi.nlm.nih.gov/books). National Library of Medicine. - [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH. - [NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules](https://osp.od.nih.gov/policies/biosafety-and-biosecurity-policy/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules). National Institutes of Health. - [Addgene Protocols](https://www.addgene.org/protocols). Addgene. - [MIQE Guidelines](https://academic.oup.com/clinchem/article/55/4/611/5631762). Clinical Chemistry. - [NIST Laboratory Programs](https://www.nist.gov/laboratories). National Institute of Standards and Technology. - [Determination of Protein Molecular Weights on SDS-PAGE.](https://pubmed.ncbi.nlm.nih.gov/30426411). Methods in molecular biology (Clifton, N.J.), 2019. - [Blue native PAGE.](https://pubmed.ncbi.nlm.nih.gov/17406264). Nature protocols, 2006. - [Native polyacrylamide gels.](https://pubmed.ncbi.nlm.nih.gov/22585476). Methods in molecular biology (Clifton, N.J.), 2012. - [Mobilization in two-step capillary isoelectric focusing: Concepts assessed by computer simulation.](https://pubmed.ncbi.nlm.nih.gov/38115749). Electrophoresis, 2024. - [Isoelectric focusing and two-dimensional gel electrophoresis.](https://pubmed.ncbi.nlm.nih.gov/19892190). Methods in enzymology, 2009. > This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.