[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$f99NxdEkIhxLWdcZTTWlNYFISPFf0jq_Et6OdgLFJJUc":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":238},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":36,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":39,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"body":43,"faq":44,"tags":45,"related":47},"capillary-electrophoresis","Capillary Electrophoresis: Principle and Application",null,"Ashma Shrestha","2022-12-16","2026-07-05",false,"lab-equipment","Electrophoresis is a method of separation where the movement of ions under the influence of electricity helps separate components. There are various types of electrophoresis. Gel electrophoresis, cellular acetate electrophoresis, and capillary electrophoresis are some of the commonly used types of electrophoresis.\n\n**Capillary electrophoresis is a liquid-based separation technique that uses a capillary as a separation channel under the influence of an electric field. It is an analytical technique where the electroosmotic flow helps separately charged ions. It is applicable in various areas like analysis of chemical substances, DNA analysis, identifying specific proteins, and separating coenzymes.**\n\n## Principle of Capillary Electrophoresis\n\nThe velocity of the analyte when migrating under the influence of an electric field of intensity “E” is given by the analyte’s electrophoretic mobility and the buffer’s electroosmotic mobility inside the capillary.\n\nThe electrophoretic mobility of solute depends on the different characteristics of the solute, like electric charge, molecular size, and shapes, and the properties of the buffer, like electrolyte’s ionic strength, pH, viscosity, and additives. The following equation gives the electrophoretic velocity (Vep) of the solute:\n\nVep= μepE = (q\u002F6πηr) (V\u002FL) where η is the viscosity of the electrolyte solution, V is the applied voltage, L= the length of the capillary, r is the Stoke’s radius of the solute, μep is the electrophoretic mobility of the solute, and q is the effective charge of the solute.\n\nWhen applied through the capillary filled with the buffer, the electric field generates a solvent flow called the electroosmotic flow inside the capillary. The velocity of which depends on the electrophoretic mobility. Electrophoretic mobility depends on the charge density of the capillary’s internal wall and the buffer’s properties. The following equation gives the electroosmotic velocity (Veo):\n\nVeo = μeoE = (ε𝜁\u002Fη) (V\u002FL) where ε= buffer’s dielectric constant, 𝜁= zeta potential of the capillary surface, η is the viscosity of the electrolyte solution, V is the applied voltage, μeo is the electrophoretic mobility, and L is the length of the capillary.\n\nThe solute’s velocity (V) is given by: V= Vep+Veo.\n\nThe electroosmotic and electrophoretic mobility of the analyte can act in the same or opposite direction based on the solute’s charge. In the case of normal capillary electrophoresis, anions will move in the opposite direction of electroosmotic flow with velocities smaller than the electroosmotic velocity. Whereas, the cations will migrate in the same direction of the electroosmotic flow with velocities higher than the electroosmotic velocity. In conditions with a fast electroosmotic rate compared to the electrophoretic rate of the solutes, the separation of both cations and anions occurs in the same run. The time is taken (t) for the solute to migrate to the detection point from the injection end of the capillary(effective capillary length) is given by:\n\nt = l\u002F Vep+Veo = l(L)\u002F V( Vep+Veo)\n\n![Capillary electrophoresis - Setup of Capillary Electrophoresis](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FCapillaryelectrophoresis.png)\n\u003Cfigcaption>Figure: Setup of Capillary Electrophoresis\u003C\u002Ffigcaption>\n\nGenerally, uncoated fused silica capillaries above pH three that have a negative charge are used where the electroosmotic flow occurs from the anode to the cathode. The electroosmotic flow must be constant for each run for good reproducibility in the migration velocity of the solutes. For some experiments, reduction or suppression of electroosmotic glow by changing the inner wall of the capillary, the concentration, composition, or pH of the buffer solution might be necessary.  \n\nAfter adding the sample, an independent zone of each analyte ion of the sample forms. The zone formation is due to the migration within the background electrolyte. The spreading of each solute band occurs as a result of different phenomena. Under ideal conditions, the only phenomenon contributing to the zone’s broadening is molecular diffusion of the solute inside the capillary. Here, the efficiency of the zone is given as the number of theoretical plates (N), which is given by:\n\nN= (μep+μeo) (Vl)\u002F 2DL, where D is the molecular diffusion coefficient of the solute buffer.\n\nIn general practice, other phenomena like the length of the injection plug, detector cell size, unleveled buffer reservoirs, mismatched conductivity between sample and buffer, sample adsorption onto the capillary wall, and heat dissipation, play a significant role in band dispersion. The separation between two bands is gained by modifying the analytes’ electrophoretic mobility, the electroosmotic mobility induced in the capillary, and increasing each analyte’s efficiency for the band as follows;\n\nRs= N(μepb-μepa)\u002F 4(μaep+μeo); where μepa and μepb= the two analytes’ electrophoretic mobilities, μaep is the average electrophoretic mobility of the two analytes calculated by: μaep= ½ ( μepb+μepa).\n\n## Instruments Used in Capillary Electrophoresis\n\n1. **Power supply:** Capillary electrophoresis requires a high voltage controllable direct current power supply.\n2. **Buffer reservoirs:** Capillary electrophoresis requires two buffer reservoirs held at the same level that contain specified anodic and cathodic solutions.\n3. **Electrodes:** CE needs two electrodes (cathode and anode) immersed in the buffer reservoirs connected to the power supply.\n4. **Capillary:** A capillary made up of fused silica, usually with a diameter of fewer than 100 microns.\n5. **Viewing window:** CE requires an optical viewing window that is aligned to the detector.\n6. **Injection system:** CE requires a suitable injection system for adding sample and buffer into the capillary. The method of injection can be automated for precision. The common mode of injecting samples is gravity, pressure or vacuum, or electrokinetic.\n7. **Detector:** CE requires a detector for monitoring the amount of substance that passes through the capillary at a given time based on conductimetric, fluorimetry, absorption spectrophotometry (UV and visible), amperometric, or mass spectrometric detection.\n8. **Thermostatic system:** CE requires a thermostatic system for maintaining a constant temperature inside the capillary.\n9. **Recorder:** CE requires a recorder that records the data after the completion of electrophoresis.\n10. **Suitable integrator or computer:** CE also needs the right integrator or computer to convert data digitally.  \n\n## Types of Capillary Electrophoresis Method\n\nThere are six types of capillary electrophoresis methods commonly used; CZE (capillary zone electrophoresis), CGE (capillary gel electrophoresis), Micellar electro kinetic capillary chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP).\n\n### Capillary zone electrophoresis (CZE)\n\nThe separation in this type of electrophoresis requires a capillary with only a buffer without any anticonvective medium. The analytes are separated into bands whose velocity depends on the electrophoretic mobility and electroosmotic flow. The electroosmotic flow moves toward the cathode, and when the polarity has reversed, the analytes with electroosmotic mobilities higher than the electroosmotic force will pass the outlet. The coated capillaries help increase the separation capacity of the substances that adhere to the fused-silica surfaces. The CZE is applicable in analyzing small to large (\u003C2000 molecular weight to \u003C100,000 molecular weight). It is a very efficient form of capillary electrophoresis.****\n\n### Capillary gel electrophoresis (CGE)\n\nThe separation in capillary gel electrophoresis occurs in a capillary filled with a gel acting as a molecular sieve quite similar to [gel electrophoresis](\u002Fagarose-gel-electrophoresis\u002F). Molecules are separated according to molecular size. The smaller molecules move more freely through the gel network; hence these migrate faster than larger molecules. Capillary gel electrophoresis is used to separate proteins, DNA fragments, and other biological macromolecules with similar charge-to-mass ratios based on their molecular size. ****\n\n### Micellar electrokinetic capillary chromatography (MEKC)\n\nThe separation occurs in an electrolytic solution that consists of a surfactant with a concentration above the critical micellar concentration (CMC). This technique is a hybrid of electrophoresis and chromatography. The method is applicable to both neutral and charged solutes. The solute molecules get distributed between the pseudo-stationary phase made up of micelles and aqueous buffer based on the solute’s partition coefficient. The commonly used surfactant in MKEC is sodium dodecyl sulfate, cetyl trimethyl, and ammonium salts. In neutral and alkaline pH, the electroosmotic flow is strong, and the separation buffer ions move toward the direction of the cathode.\n\nIn case sodium dodecyl sulfate (SDS) is used as a surfactant, the anionic micelle’s electrophoretic migration is in the order of the anode, slowing down the overall micelle migration velocity in the bulk flow of the electrolytic solution. Suppose neutral solutes are present; the analyte partitions between the aqueous buffer and the micelle and lacks electrophoretic mobility. In such cases, the analyte’s migration velocity depends on the partition coefficient between the aqueous buffer and the micelle. Because of this, the separation in neutral and weakly ionized solutes in MKEC is chromatographic. In the case of charged solutes, migration velocity depends on the electrophoretic mobility of the solute without a micelle and the partition coefficient between the aqueous buffer and the micelle.\n\n### Capillary electrochromatography (CEC)\n\nCapillary electrochromatography is a hybrid separation technique that uses the principle of both capillary electrophoresis and chromatography, especially [high-performance liquid chromatography (HPLC)](\u002Fhplc-high-performance-liquid-chromatography\u002F). Analytes separate based on the differences in the partition ratio between the mobile and stationary phase or due to electrophoretic mobility. The mobile phase is run across the chromatographic bed with the help of electroosmotic force instead of pressure. CEC is more advantageous than HPLC and capillary electrophoresis. It applies to enantiomeric separation, separating amino acids, proteins, peptides, and carbohydrates. It is mainly used in pharmaceutical industries to identify acidic and basic drugs and in the industrial sector that involves analyzing polymers. ****\n\n### Capillary isoelectric focusing (CIEF)\n\nThe charged molecules migrate due to the electric field in the isoelectric focusing in a pH gradient obtained from the ampholytes having pI (isoelectric point) value in a wide range that is dissolved in the separation buffer. There are three steps in CIEF; loading, focusing, and mobilization. The introduction of the ampholytes and separation buffer is loaded in the capillary in the loading step. In the focusing step, the voltage is provided in the capillary, causing the ampholytes to move to their respective electrode based on their net charges and creating a pH gradient where the molecules migrate until they reach a pH corresponding to their pI (isoelectric point). The mobilization can be required for detection, which is obtained by either focusing under the influence of electroosmotic flow, applying positive pressure after focusing, or adding salt to the anode or cathode reservoir, depending on the direction of mobilization.\n\n### Capillary isotachophoresis (CITP)\n\nThe technique was developed to separate the serum lipoproteins. Compared to other electrophoretic processes, it has negligible molecular sieve effects, requires no gel casting, applicable for whole serum, and can easily differentiate lipoprotein subfractions. Here, the total serum lipoproteins are pre-stained in a free-flow capillary system (with an internal diameter of 0.5 mm) having a discontinuous buffer system for 30 minutes at 4℃ before starting the process. The lipoproteins are separated into HDL (high-density lipoproteins) and LDL (low-density lipoproteins) based on electrophoretic mobility. The method is commonly used in clinical laboratories for determining the lipoprotein level in human serum.\n\n## Application of Capillary Electrophoresis\n\nCapillary electrophoresis is commonly applied in various fields. Analyzing food contents, genetic differentiation, and clinical diagnosis are typical applications of capillary electrophoresis. The following is a detailed explanation of the area of applications of capillary electrophoresis:\n\n1. **Food technology:**Capillary electrophoresis is applied to analyze different food, like beverages and fermented food, to detect flavonoids, vitamins, carbohydrates, proteins, pigments, and color. CE is also applicable for detecting DNA and RNA from bacteria and viruses, which indicates contamination.\n2. **Molecular analysis:** The DNA, RNA, and amino acids are detected using capillary electrophoresis, especially in the microbiology laboratory and forensic science.\n3. **Disease diagnosis:** Lipid profile analysis using CITP is critical for detecting cholesterol levels in clinical laboratories. Likewise, the human serum’s analysis of vitamins and minerals is performed using capillary electrophoresis.  \n4. **Environmental monitoring:** Capillary electrophoresis has been applied to track inorganic ions in rivers and water. (4) Likewise, nano-capillary electrophoresis is applied to detect environmental pollutants at the nano level. (5)\n5. **Pharmaceutical laboratories:** Capillary electrophoresis is used in the analysis of small molecules in drugs during preparatory stages and standard solutions. (6)\n\n## Advantages of Capillary Electrophoresis\n\nCapillary electrophoresis has many advantages over other methods of [electrophoresis](\u002Felectrophoresis-principles-types-and-uses\u002F) and [chromatography](\u002Fchromatography-an-overview\u002F) due to the use of tiny capillaries for separating. The following is a detailed explanation of the benefits of capillary electrophoresis:  \n\n1. **Highly efficient:** Heat dissipation occurs efficiently due to using a small diameter of the capillary. Some articles claim the efficiency of capillary electrophoresis is quite similar to that of liquid chromatography. (7)\n2. **Less time-consuming:** If the person is familiar with computer software, it takes no time to read the result of separation. Likewise, the entire process takes only one hour, from setup to separation. So, capillary electrophoresis carries more advantages as compared to other separation techniques.\n3. **Wide area of application:** Capillary electrophoresis is used in analyzing macromolecules like proteins, DNA, different drugs, and RNA. So, CE is applied widely from pharmaceutical to environmental monitoring.  \n4. **Has the possibility of automation:** The injection method, data analysis, buffer, and sample uploading steps can be automated in capillary electrophoresis.  \n\n## Disadvantages of Capillary Electrophoresis\n\nAlthough capillary electrophoresis is highly advantageous, it has some disadvantages, which are as follows:\n\n1. **Changing capillaries is challenging:** Due to the size of the capillaries, removing and replacing capillaries can be challenging at times. So, expertise is required for changing capillaries to experiment.\n2. **Less robust:** Small changes like the size of capillaries and choice of analytes can be crucial in capillary electrophoresis. So CE is less robust than other methods.\n\n**References**\n\n1. *Capillary electrophoresis*. (n.d.). Retrieved December 16, 2022, from [https:\u002F\u002Fwww.usp.org\u002Fsites\u002Fdefault\u002Ffiles\u002Fusp\u002Fdocument\u002Fharmonization\u002Fbiotechnology\u002Fharmonization-september-2019-m859.pdf](https:\u002F\u002Fwww.usp.org\u002Fsites\u002Fdefault\u002Ffiles\u002Fusp\u002Fdocument\u002Fharmonization\u002Fbiotechnology\u002Fharmonization-september-2019-m859.pdf)\n2. Borst, C., Belal, F., & Holzgrabe, U. (2013). Possibilities and limitations of capillary electropherosis in pharmaceutical analysis. *Die Pharmazie*, *68*(7), 526–530.\n3. Robert, F., Bouilloux, J. P., & Denoroy, L. (1991). L’électrophorèse capillaire: principe et applications [Capillary electrophoresis: principle and applications]. *Annales de biologie clinique*, *49*(3), 137–148.\n4. Sirén, H., & Väntsi, S. (2002). Environmental water monitoring by capillary electrophoresis and result comparison with solvent chemistry techniques. *Journal of chromatography. A*, *957*(1), 17–26. [https:\u002F\u002Fdoi.org\u002F10.1016\u002Fs0021-9673(02)00218-2](https:\u002F\u002Fdoi.org\u002F10.1016\u002Fs0021-9673(02)00218-2)\n5. Ali, I., Alharbi, O. M. L., & Marsin Sanagi, M. (2016). Nano-capillary electrophoresis for environmental analysis. *Environmental chemistry letters*, *14*(1), 79–98. [https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs10311-015-0547-x](https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs10311-015-0547-x)\n6. Ali, I., Alharbi, O. M. L., & Marsin Sanagi, M. (2016). Nano-capillary electrophoresis for environmental analysis. *Environmental chemistry letters*, *14*(1), 79–98. [https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs10311-015-0547-x](https:\u002F\u002Fdoi.org\u002F10.1007\u002Fs10311-015-0547-x)\n7. Masár, M., Hradski, J., Schmid, M. G., & Szucs, R. (2020). Advantages and Pitfalls of Capillary Electrophoresis of Pharmaceutical Compounds and Their Enantiomers in Complex Samples: Comparison of Hydrodynamically Opened and Closed Systems. *International journal of molecular sciences*, *21*(18), 6852. [https:\u002F\u002Fdoi.org\u002F10.3390\u002Fijms21186852](https:\u002F\u002Fdoi.org\u002F10.3390\u002Fijms21186852)",[],[46],"electrophoresis",[48,87,124,160,168,206],{"slug":49,"title":50,"description":51,"seoTitle":37,"seoDescription":37,"author":52,"createdDate":53,"lastUpdatedDate":54,"draft":41,"category":42,"image":37,"faq":55,"tags":86},"polyacrylamide-gel-electrophoresis-page","Polyacrylamide Gel Electrophoresis (PAGE): Principle and Procedure","Polyacrylamide gel electrophoresis (PAGE) separates proteins by size. Learn why SDS is added, why the gel has a stacking and a resolving layer, how to choose the acrylamide percentage, and how SDS-PAGE underpins the Western blot.","Samikshya Acharya","2023-03-19","2026-07-11",[56,59,62,65,68,71,74,77,80,83],{"question":57,"answer":58},"Why is SDS added in SDS-PAGE?","SDS is an anionic detergent that unfolds the protein and binds along the polypeptide chain at a roughly constant ratio of about one SDS molecule per two amino acid residues. This gives every protein a negative charge proportional to its length, so that charge per unit mass becomes the same for all proteins. With the charge variable removed, migration depends on size alone, and the distance a band travels can be read directly as a molecular weight.",{"question":60,"answer":61},"Why does an SDS-PAGE gel have two layers?","The upper stacking gel (pH 6.8, large pores) does no separating. Its job is to compress proteins scattered throughout the depth of the loading well into a single thin disc, using a discontinuous buffer system in which slow-moving glycine trails and fast-moving chloride leads, sandwiching the proteins between them. When the disc reaches the lower resolving gel (pH 8.8, small pores), glycine ionizes and overtakes, the sandwich collapses, and all proteins begin separating from the same starting line. Without a stacking gel, every band would be a smear.",{"question":63,"answer":64},"What is the difference between native PAGE and SDS-PAGE?","SDS-PAGE denatures the protein with SDS and a reducing agent, so separation is by size alone and the protein is no longer functional. Native PAGE uses neither, so the protein retains its fold, subunits, and intrinsic charge, and separation depends on charge, size, and shape together. Use SDS-PAGE to measure the size of a polypeptide chain, and native PAGE to study a protein that must remain active or intact.",{"question":66,"answer":67},"How do I choose the acrylamide percentage?","Match the pore size to the size of your target. A low-percentage gel (4 to 8%) has large pores and resolves large proteins, while small proteins run straight through. A high-percentage gel (12 to 20%) has small pores that resolve small proteins sharply while large proteins barely enter the gel. Higher percentage does not mean better resolution in general; it means better resolution of smaller molecules.",{"question":69,"answer":70},"Which direction do proteins move in SDS-PAGE, and why?","Toward the anode, the positive electrode. SDS coats every protein with a strong negative charge, so all proteins become anions and are attracted to the positive electrode. This is why SDS-PAGE gels are run vertically with the anode at the bottom.",{"question":72,"answer":73},"What is the role of β-mercaptoethanol, and how is it different from SDS?","They denature different things. SDS unfolds the polypeptide chain and coats it with charge, but it cannot break covalent disulfide bonds. β-mercaptoethanol is a reducing agent that cleaves those bonds, separating proteins into their individual polypeptide chains. Immunoglobulin G, for example, runs as a single band of about 150 kDa without a reducing agent, and splits into heavy chains of about 50 kDa and light chains of about 25 kDa when β-mercaptoethanol is added.",{"question":75,"answer":76},"What do APS and TEMED do?","Ammonium persulfate (APS) is the free-radical initiator that starts acrylamide polymerization, and TEMED is the catalyst that accelerates radical formation from APS. Both are added immediately before the gel is poured, because polymerization begins as soon as they are mixed in. Oxygen inhibits polymerization, which is why water-saturated isobutanol is layered over the resolving gel to exclude air.",{"question":78,"answer":79},"Is polyacrylamide gel toxic?","Unpolymerized acrylamide monomer is a potent neurotoxin and a probable human carcinogen, and it is absorbed through the skin, so the powder and the unset gel solution must be handled with gloves. Once polymerized, the gel itself is far less hazardous, but it may contain traces of residual monomer, so gloves are worn when handling gels as well.",{"question":81,"answer":82},"What is the tracking dye in SDS-PAGE?","Bromophenol blue, not bromothymol blue. It is a small, fast-migrating dye that runs ahead of nearly all proteins, marking the dye front. It does not stain the proteins. When the dye front approaches the bottom of the gel, the run is stopped so that the smallest proteins do not run off the end.",{"question":84,"answer":85},"How is SDS-PAGE related to the Western blot?","SDS-PAGE is the first step of a Western blot. Proteins are separated by molecular weight on the gel, transferred to a membrane, and then probed with antibodies. Because separation is by size alone, the position of a band on the membrane identifies the protein. This is why HIV proteins carry names such as p24, gp41, and gp120: the numbers are the molecular weights in kilodaltons at which those proteins resolve.",[46],{"slug":88,"title":89,"description":90,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":91,"lastUpdatedDate":54,"draft":41,"category":42,"image":37,"faq":92,"tags":123},"cellulose-acetate-electrophoresis","Cellulose Acetate Electrophoresis: Principle and Application","Cellulose acetate electrophoresis separates serum proteins and hemoglobin variants by net charge at alkaline pH. Learn the principle, why Hb S co-migrates with Hb D and Hb G, and why a sickle cell result must be confirmed at acid pH.","2022-12-25",[93,96,99,102,105,108,111,114,117,120],{"question":94,"answer":95},"What is the principle of cellulose acetate electrophoresis?","Proteins are separated on a cellulose acetate membrane soaked in an alkaline buffer, usually pH 8.4 to 8.6. Because the membrane's pores are large compared with a protein, it does almost no sieving, so separation depends on net charge rather than on size. At this alkaline pH the buffer is above the isoelectric point of every serum protein, so all carry a net negative charge and migrate toward the anode. The greater the net negative charge, the further the protein travels.",{"question":97,"answer":98},"Does cellulose acetate electrophoresis separate proteins by their isoelectric point?","No. A protein at its isoelectric point carries no net charge and does not migrate at all. Separation occurs because the buffer pH is held above the isoelectric point of every protein, so all are negatively charged and all move. The isoelectric point determines how negatively charged a protein is at the working pH, and therefore how fast it travels, but the separation does not occur at the isoelectric point. Separating molecules at their isoelectric points is a different technique, called isoelectric focusing.",{"question":100,"answer":101},"Why is cellulose acetate used for hemoglobin rather than a gel?","Hemoglobin variants such as Hb A and Hb S differ by a single amino acid and are essentially identical in size. A sieving matrix such as agarose or polyacrylamide separates by size and therefore cannot distinguish them. The large pores of cellulose acetate mean the membrane contributes almost nothing mechanically, so the separation is driven purely by the charge difference that the amino acid substitution creates.",{"question":103,"answer":104},"In what order do hemoglobins migrate on alkaline cellulose acetate?","Toward the anode, fastest first: Hb A, then Hb F, then Hb S, then Hb C. In Hb S, the negatively charged glutamate at position 6 of the beta chain is replaced by a neutral valine, reducing the net negative charge, so Hb S lags behind Hb A. In Hb C that same glutamate is replaced by a positively charged lysine, so Hb C lags further still.",{"question":106,"answer":107},"Can sickle cell disease be diagnosed from an alkaline cellulose acetate strip alone?","No. At alkaline pH, Hb S co-migrates with Hb D, Hb G, and Hb Lepore, and Hb C co-migrates with Hb E, Hb O-Arab, and Hb A2. A band at the S position identifies a charge, not a hemoglobin. Confirmation requires electrophoresis on citrate agar at acid pH, around 6.0 to 6.2, where Hb S separates from Hb D and Hb G, supported by a sickle solubility test.",{"question":109,"answer":110},"How do you distinguish sickle cell trait from sickle cell disease on the strip?","Look for the Hb A band. Sickle cell trait shows two bands, with Hb A denser than Hb S in roughly a 60 to 40 ratio. Sickle cell disease shows no Hb A band at all, a dense Hb S band, and often a raised Hb F. The absence of Hb A is what makes the diagnosis. A recent blood transfusion can introduce a donor-derived Hb A band and obscure this.",{"question":112,"answer":113},"Why are newborn screening results written as FS or FAS?","Hemoglobin bands in newborn screening are reported in descending order of abundance, and a newborn still produces mostly fetal hemoglobin. FA is a normal newborn, FAS indicates sickle cell trait, and FS indicates sickle cell disease, because no Hb A is present.",{"question":115,"answer":116},"Why do gamma globulins appear behind the point of application?","Because of electroendosmosis. Fixed negative charges on the cellulose acetate attract cations from the buffer, and the resulting bulk flow of buffer moves toward the cathode. This flow opposes the anodal migration of the proteins. Albumin carries enough net charge to overcome it easily, but the gamma globulins, which are the least negatively charged fraction, are dragged back to or slightly past the origin.",{"question":118,"answer":119},"How are the bands visualized on a cellulose acetate strip?","The strip is stained with a protein dye such as Ponceau S, Amido Black, or Coomassie brilliant blue, then destained in dilute acetic acid to remove background. It is then cleared in a methanol or acetic acid mixture, which turns the cellulose acetate optically transparent, allowing the strip to be scanned directly in a densitometer for quantification and stored as a permanent record. Where the analytes are enzymes, a zymogram may be used instead, revealing bands by their enzymatic activity.",{"question":121,"answer":122},"Is cellulose acetate electrophoresis still used?","Yes. In well-resourced laboratories, HPLC and capillary electrophoresis have largely replaced it for hemoglobin variant analysis, because they are automated, quantitative, and resolve variants that co-migrate at alkaline pH. Cellulose acetate remains widely used where those instruments are unavailable, and it retains the advantages of being simple, rapid, and inexpensive.",[46],{"slug":125,"title":126,"description":127,"seoTitle":37,"seoDescription":37,"author":128,"createdDate":129,"lastUpdatedDate":130,"draft":41,"category":42,"image":37,"faq":131,"tags":159},"electrophoresis-principles-types-and-uses","Electrophoresis: Principles, Types, and Uses","Electrophoresis separates charged molecules such as proteins and DNA by moving them through a gel in an electric field. Learn the principle, the factors that control mobility, the main types, and how serum protein electrophoresis detects multiple myeloma.","Srijana Khanal","2022-07-13","2026-07-10",[132,135,138,141,144,147,150,153,156],{"question":133,"answer":134},"What is the basic principle of electrophoresis?","Charged molecules placed in an electric field migrate toward the electrode of opposite charge. Negatively charged molecules (anions) move toward the positive anode, and positively charged molecules (cations) move toward the negative cathode. Each molecule travels at a speed set by its electrophoretic mobility, which depends on its net charge, its size and shape, and the viscosity and pore size of the medium. Molecules separate only if their mobilities differ.",{"question":136,"answer":137},"Why does DNA always move toward the anode?","DNA carries a phosphate backbone that remains negatively charged at any pH used in the laboratory. Because it is always an anion, it is always attracted to the positive anode. Its charge-to-mass ratio is also nearly constant regardless of fragment length, which is why DNA fragments separate essentially by size alone.",{"question":139,"answer":140},"Which way does a protein move in electrophoresis?","It depends on the buffer pH relative to the protein's isoelectric point (pI). Above its pI the protein is net negative and moves toward the anode. Below its pI it is net positive and moves toward the cathode. At exactly its pI, its net charge is zero and it does not migrate.",{"question":142,"answer":143},"Why is electrophoresis called an incomplete form of electrolysis?","In electrolysis, ions travel all the way to the electrode and undergo discharge there. In electrophoresis the electric field is switched off while the molecules are still in transit, so they never reach the electrode. What matters is not the reaction at the electrode but how far each molecule traveled, because that distance is the separation.",{"question":145,"answer":146},"What is the difference between zone and moving boundary electrophoresis?","In zone electrophoresis the sample is applied as a narrow zone on a supporting medium such as paper, cellulose acetate, or a gel, and components resolve into discrete bands. In moving boundary electrophoresis the separation occurs in free solution with no supporting medium, and the components appear as moving boundaries rather than distinct bands. The classical example of the latter is the Tiselius apparatus.",{"question":148,"answer":149},"What are the main factors affecting electrophoretic mobility?","Inherent factors include the net charge of the molecule, its charge density, its molecular weight, and its size and shape. External factors include the applied voltage, current and power, the pore size and viscosity of the supporting medium, the temperature, and the pH of the buffer, which determines the net charge on ampholytes such as proteins.",{"question":151,"answer":152},"How is electrophoresis used to diagnose multiple myeloma?","Serum protein electrophoresis separates serum proteins into albumin and the alpha, beta, and gamma globulin fractions. Normal gamma globulins are produced by thousands of plasma cell clones with slightly different mobilities, so they form a broad band. In multiple myeloma a single malignant clone produces one identical immunoglobulin, and these identical molecules migrate together to produce a sharp, narrow monoclonal (M) band in the gamma region.",{"question":154,"answer":155},"Does electrophoresis separate molecules by size or by charge?","By both, because mobility depends on the ratio of net charge to size. SDS-PAGE deliberately removes the charge variable by coating every protein with a uniform negative charge proportional to its length, so that separation depends on size alone. Native gels, in contrast, separate molecules on the basis of charge and size together.",{"question":157,"answer":158},"Why is a larger pore size not always better?","Larger pores impede migration less, so molecules travel faster, but small molecules pass through almost unhindered and are therefore poorly resolved. The gel concentration is chosen to match the size range of interest: a low-percentage gel resolves large fragments, and a high-percentage gel resolves small ones.",[46],{"slug":161,"title":162,"description":163,"seoTitle":37,"seoDescription":37,"author":164,"createdDate":165,"lastUpdatedDate":54,"draft":41,"category":42,"image":37,"faq":166,"tags":167},"pulsed-field-gel-electrophoresis-pfge","Pulsed-Field Gel Electrophoresis (PFGE): Steps, Applications","Pulsed-field gel electrophoresis (PFGE) separates DNA fragments up to 10 Mb by switching the electric field between directions, forcing large molecules to reorient. Learn why pulsing works, the plug-based steps, and why PFGE was the gold standard for outbreak fingerprinting.","Acharya Tankeshwar","2019-09-16",[],[46],{"slug":169,"title":170,"description":171,"seoTitle":37,"seoDescription":37,"author":164,"createdDate":172,"lastUpdatedDate":173,"draft":41,"category":42,"image":37,"faq":174,"tags":205},"agarose-gel-electrophoresis","Agarose Gel Electrophoresis: Principle, Procedure, Results","Agarose gel electrophoresis separates DNA fragments from 100 bp to 25 kb by size. Learn the principle, how to cast and run a gel, why supercoiled plasmid runs faster than linear DNA, and how to read a PCR gel against a DNA ladder.","2019-09-13","2026-07-17",[175,178,181,184,187,190,193,196,199,202],{"question":176,"answer":177},"Does agarose gel electrophoresis separate DNA by size or by charge?","By size. Charge determines the direction of travel, because DNA's phosphate backbone is negatively charged and every fragment therefore migrates toward the anode. But DNA has a uniform charge-to-mass ratio, so every fragment experiences the same pull per unit mass, and charge separates nothing. The sieving action of the agarose mesh does all the separating, which is why migration distance reports fragment size.",{"question":179,"answer":180},"Why does DNA move toward the anode?","The phosphate backbone of DNA carries a negative charge at every pH used in the laboratory, making DNA an anion. Anions are attracted to the positive electrode, which is the anode. Unlike proteins, DNA has no isoelectric point to consider, so it always migrates in the same direction.",{"question":182,"answer":183},"What size range can agarose gel electrophoresis resolve?","Roughly 100 base pairs to 25 kilobases, depending on the agarose concentration. Fragments smaller than about 100 bp are better resolved by polyacrylamide gel electrophoresis, and fragments larger than about 25 kb require pulsed-field gel electrophoresis.",{"question":185,"answer":186},"Why does my plasmid preparation show three bands on the gel?","Because a circular plasmid exists in three conformations, and the gel separates by effective size rather than base-pair count. Supercoiled plasmid is tightly wound and compact, so it migrates fastest. Open circular (nicked) plasmid is a relaxed floppy loop with a large effective radius, so it snags in the mesh and migrates slowest. Linear plasmid runs in between. All three contain the same number of base pairs. Three bands from one preparation is normal, not a sign of contamination.",{"question":188,"answer":189},"How do I choose the agarose concentration?","Match the pore size to the fragment size. A low-percentage gel (around 0.5 to 0.8%) has large pores and resolves large fragments, while small fragments run through almost unimpeded. A high-percentage gel (1.5 to 2%) has small pores that resolve small fragments sharply while holding large fragments near the well. Higher percentage does not mean better resolution in general, only better resolution of smaller fragments.",{"question":191,"answer":192},"What is the difference between TAE and TBE buffer?","TAE (Tris-acetate-EDTA) has a lower buffering capacity and will exhaust during long runs, but it resolves large fragments well and the DNA recovered from a TAE gel is clean enough for downstream enzymatic work. TBE (Tris-borate-EDTA) has a much higher buffering capacity and gives sharper resolution of small fragments, but borate inhibits many enzymes and carries over into extracted DNA. Use TAE if you plan to cut the band out and use the DNA, and TBE if you only need to visualize it.",{"question":194,"answer":195},"What is a DNA ladder and why is it needed?","A DNA ladder is a mixture of DNA fragments of known sizes, run in a lane alongside the samples. It converts the height of a band into a number of base pairs. Without a ladder, the position of a band carries no information, because migration distance depends on the gel percentage, the voltage, and the run time.",{"question":197,"answer":198},"My PCR gel shows no band in the patient lane. Is that a negative result?","Only if the positive control produced a band. If the positive control is also blank, the reaction itself failed and the patient's lane carries no information. Likewise, if the negative control shows a band, the run is contaminated and no lane on that gel can be trusted. The controls are always read before the patient's sample.",{"question":200,"answer":201},"What do the dyes in the loading buffer do?","Loading dye serves three purposes. Glycerol makes the sample dense enough to sink to the bottom of the well instead of drifting into the buffer. The colour makes loading easier to see. And the tracking dyes migrate at predictable rates, marking how far the run has progressed. In a 1% agarose gel, bromophenol blue migrates at approximately the position of a 300 to 500 bp fragment and xylene cyanol at approximately 4 kb.",{"question":203,"answer":204},"Is ethidium bromide dangerous, and what can be used instead?","Ethidium bromide intercalates into DNA and is a suspect mutagen and carcinogen, so it requires gloves and regulated disposal. Safer alternatives include SYBR Gold and SYBR Green, which are highly sensitive but expensive, and methylene blue or crystal violet, which are much safer but considerably less sensitive. A separate hazard is the short-wave ultraviolet light used to visualize ethidium bromide, which nicks DNA. If the band is to be excised for cloning, use a long-wave ultraviolet or blue-light transilluminator.",[46],{"slug":207,"title":208,"description":209,"seoTitle":37,"seoDescription":37,"author":164,"createdDate":210,"lastUpdatedDate":54,"draft":41,"category":42,"image":37,"faq":211,"tags":236},"serologic-methods-counterimmunoelectrophoresis-cie","Counterimmunoelectrophoresis (CIE): Principle, Procedure, Uses","Counterimmunoelectrophoresis (CIE) drives antigen and antibody toward each other in an agarose gel, producing a precipitin line within an hour. Learn the principle, why a neutral antibody migrates at all, the procedure, and why pneumococcal serotypes 7 and 14 give false negatives.","2012-09-29",[212,215,218,221,224,227,230,233],{"question":213,"answer":214},"What is the principle of counterimmunoelectrophoresis?","Antigen and antibody are driven toward each other through an agarose gel in an alkaline buffer, and where they meet in optimal proportions they form a visible precipitin line. They move for different reasons. Bacterial capsular antigens are acidic, so they carry a net negative charge at pH 8.4 and migrate toward the anode. Antibodies carry almost no net charge at that pH, but they are swept toward the cathode by electroendosmosis, the bulk flow of buffer through the negatively charged agarose. The result is that the two travel in opposite directions along the same line and collide between the wells.",{"question":216,"answer":217},"If antibodies are electrically neutral, how do they move in CIE?","They do not move because of the electric field acting on them directly. They move because the liquid inside the gel is moving. Agarose carries fixed negative charges that attract cations from the buffer. When the current is applied, those cations migrate toward the cathode and drag hydrating water with them, so the whole buffer phase flows cathodally. This bulk flow, called electroendosmosis, carries the near-neutral antibody toward the cathode. The strongly negative antigen swims against this flow and still reaches the anode.",{"question":219,"answer":220},"Why is it called counterimmunoelectrophoresis?","Because the antigen and antibody migrate counter to one another, in opposite directions along the same axis, so that they are forced to meet. In ordinary electrophoresis everything in the gel migrates in the same direction. An older name for the technique, immunoelectroosmophoresis, describes the mechanism more literally, since electroosmosis is what moves the antibody.",{"question":222,"answer":223},"Which well should the antigen go into?","The cathodal well. The antigen migrates toward the anode, so it must start on the cathodal side to have somewhere to travel. The antibody is carried toward the cathode, so it must start in the anodal well. Each reactant begins at the electrode it is moving away from. If the wells are loaded the other way round, the two reactants migrate apart and no precipitin line can form regardless of how much antigen is present.",{"question":225,"answer":226},"How is CIE different from the Ouchterlony method?","The chemistry is identical. Both rely on antigen and antibody meeting in optimal proportions to form a precipitin line in agar. The difference is that Ouchterlony relies on passive diffusion, which sends the reactants outward in all directions and takes twenty-four to forty-eight hours. CIE applies an electric current that drives them straight toward each other along one line, giving a result in thirty to sixty minutes and detecting roughly ten times less antigen.",{"question":228,"answer":229},"How is counterimmunoelectrophoresis different from immunoelectrophoresis?","They are separate techniques with confusingly similar names. In classical immunoelectrophoresis, described by Grabar and Williams, a protein mixture is first separated by electrophoresis, and only afterwards is antiserum allowed to diffuse passively from a trough, producing precipitin arcs. Electrophoresis and immunodiffusion happen one after the other. In counterimmunoelectrophoresis they happen simultaneously, and the electric field brings the reactants together rather than separating them.",{"question":231,"answer":232},"Why detect capsular antigen rather than culture the organism?","Because capsular polysaccharide persists in cerebrospinal fluid long after antibiotics have killed the bacterium that shed it. A child given a dose of antibiotic before the lumbar puncture may have a blank Gram stain and a sterile culture while the CSF still contains abundant antigen. Antigen detection was developed for exactly this situation, and it delivers an answer in an hour rather than two days.",{"question":234,"answer":235},"Is counterimmunoelectrophoresis still used today?","Rarely in routine diagnostics. Latex particle agglutination is faster, simpler, and more sensitive, and needs no apparatus. For Cryptococcus neoformans, the cryptococcal antigen lateral flow assay is now the recommended method. For bacterial meningitis, multiplex PCR detects the organism's nucleic acid rather than its shed capsule, with far greater sensitivity. CIE survives in teaching, in some reference and veterinary laboratories, and where reagents are inexpensive and the apparatus is already available.",[46,237],"immunoassays",[239,245,251,256,259,263,268,273,276,280],{"slug":240,"name":164,"description":241,"image":242,"body":243,"postCount":244},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",432,{"slug":246,"name":38,"description":247,"image":248,"body":249,"postCount":250},"ashma-shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":252,"name":253,"description":254,"image":37,"body":37,"postCount":255},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":257,"name":52,"description":254,"image":37,"body":37,"postCount":258},"samikshya-acharya",20,{"slug":260,"name":261,"description":254,"image":37,"body":37,"postCount":262},"alisha-tripathi","Alisha Tripathi",6,{"slug":264,"name":265,"description":266,"image":37,"body":37,"postCount":267},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":269,"name":270,"description":271,"image":37,"body":37,"postCount":272},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":274,"name":128,"description":254,"image":37,"body":37,"postCount":275},"srijana-khanal",18,{"slug":277,"name":278,"description":271,"image":37,"body":37,"postCount":279},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":281,"name":282,"description":254,"image":37,"body":283,"postCount":284},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]