Gel electrophoresis of proteins

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Proteins separated by SDS-PAGE, Coomassie brilliant blue staining Coomassie3.jpg
Proteins separated by SDS-PAGE, Coomassie brilliant blue staining

Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely agarose or polyacrylamide. Variants of gel electrophoresis include SDS-PAGE, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting or immunoblotting to give additional information about a specific protein. [1]

Contents

Denaturing gel methods

SDS-PAGE

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques to separate proteins according to their electrophoretic mobility (a function of the molecular weight of a polypeptide chain) while in the denatured (unfolded) state. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. [2]

SDS is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides. When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform charge density, that is same net negative charge per unit length. The electrophoretic mobilities of these proteins will be a linear function of the logarithms of their molecular weights. [3]

Native gel methods

Native gels, also known as non-denaturing gels, analyze proteins that are still in their folded state. Thus, the electrophoretic mobility depends not only on the charge-to-mass ratio, but also on the physical shape and size of the protein. [4]

Blue native PAGE

BN-PAGE is a native PAGE technique, where the Coomassie brilliant blue dye provides the necessary charges to the protein complexes for the electrophoretic separation. [5] [6] The disadvantage of Coomassie is that in binding to proteins it can act like a detergent causing complexes to dissociate. Another drawback is the potential quenching of chemoluminescence (e.g. in subsequent western blot detection or activity assays) or fluorescence of proteins with prosthetic groups (e.g. heme or chlorophyll) or labelled with fluorescent dyes.[ citation needed ]

Clear native PAGE

CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and membrane proteins in a polyacrylamide gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in CN-PAGE (in contrast to the charge shift technique BN-PAGE) is related to the intrinsic charge of the proteins. [7] The migration distance depends on the protein charge, its size and the pore size of the gel. In many cases this method has lower resolution than BN-PAGE, but CN-PAGE offers advantages whenever Coomassie dye would interfere with further analytical techniques, for example it has been described as a very efficient microscale separation technique for FRET analyses. [8] Additionally, as CN-PAGE does not require the harsh conditions of BN-PAGE, it can retain the supramolecular assemblies of membrane protein complexes that would be dissociated in BN-PAGE. [7]

Preparative native PAGE

The folded protein complexes of interest separate cleanly and predictably without the risk of denaturation due to the specific properties of the polyacrylamide gel, electrophoresis buffer solution, electrophoretic equipment and standardized parameters used. The separated proteins are continuously eluted into a physiological eluent and transported to a fraction collector. In four to five PAGE fractions each the different metal cofactors can be identified and absolutely quantified by high-resolution ICP-MS. The associated structures of the isolated metalloproteins in these fractions can be specifically determined by solution NMR spectroscopy. [9]

Buffer systems

Postulated migration of proteins in a Laemmli gel system A: Stacking gel, B: Resolving gel, o: sample application c: discontinuities in the buffer and electrophoretic matrix Laemmli System.png
Postulated migration of proteins in a Laemmli gel system A: Stacking gel, B: Resolving gel, o: sample application c: discontinuities in the buffer and electrophoretic matrix

Most protein separations are performed using a "discontinuous" (or DISC) buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins. These conditions provide an environment in which Kohlrausch's reactions determine the molar conductivity. As a result, SDS-coated proteins are concentrated to several fold in a thin zone of the order of 19 μm within a few minutes. At this stage all proteins migrate at the same migration speed by isotachophoresis. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. [10] [11] Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and eliminate the ion gradient and thereby the stacking effect.[ citation needed ]

A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may promote disulfide bond formation between cysteine residues in the proteins because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use. [12] [13]

SDS gradient gel electrophoresis of proteins

As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is Cl − ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl− of the gel buffer and the Gly − of the cathode buffer. Friedrich Kohlrausch found that Ohm's law also applies to dissolved electrolytes. Because of the voltage drop between the Cl− and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layers. [14] The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position. For a complete protein unstacking the polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a "stacking-gel" with a different pH is not needed. [15]

Visualization

The most popular protein stain is Coomassie brilliant blue. It is an anionic dye, which non-specifically binds to proteins. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. [16] [17]

When more sensitive method than staining by Coomassie is needed, silver staining is usually used. Silver staining is a sensitive procedure to detect trace amounts of proteins in gels, but can also visualize nucleic acid or polysaccharides. [17]

Visualization methods without using a dye such as Coomassie and silver are available on the market. [18] For example Bio-Rad Laboratories markets ”stain-free” gels for SDS-PAGE gel electrophoresis. Alternatively, reversible fluorescent dyes, such as those from Azure Biosystems such as AzureRed or Azure TotalStain Q can be used. [17] [18] [19]

Similarly as in nucleic acid gel electrophoresis, tracking dye is often used. Anionic dyes of a known electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is Bromophenol blue. This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins. [20]

Medical applications

Schematic representation of a protein electrophoresis gel. Electrophoresis.png
Schematic representation of a protein electrophoresis gel.
Serum protein electrophoresis showing a paraprotein (peak in the gamma zone) in a patient with multiple myeloma. Monoclonal gammopathy Multiple Myeloma.png
Serum protein electrophoresis showing a paraprotein (peak in the gamma zone) in a patient with multiple myeloma.

In medicine, protein electrophoresis is a method of analysing the proteins mainly in blood serum. Before the widespread use of gel electrophoresis, protein electrophoresis was performed as free-flow electrophoresis (on paper) or as immunoelectrophoresis.[ citation needed ]

Traditionally, two classes of blood proteins are considered: serum albumin and globulin. They are generally equal in proportion, but albumin as a molecule is much smaller and lightly, negatively-charged, leading to an accumulation of albumin on the electrophoretic gel. A small band before albumin represents transthyretin (also named prealbumin). Some forms of medication or body chemicals can cause their own band, but it usually is small. Abnormal bands (spikes) are seen in monoclonal gammopathy of undetermined significance and multiple myeloma, and are useful in the diagnosis of these conditions.[ citation needed ]

The globulins are classified by their banding pattern (with their main representatives):[ citation needed ]

See also

Related Research Articles

<span class="mw-page-title-main">Agarose gel electrophoresis</span> Method for separation and analysis of biomolecules using agarose gel

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.

<span class="mw-page-title-main">Gel electrophoresis</span> Method for separation and analysis of biomolecules

Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also used. For brevity, this article uses pI. The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).

<span class="mw-page-title-main">Polyacrylamide gel electrophoresis</span> Analytical technique

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

<span class="mw-page-title-main">Gel electrophoresis of nucleic acids</span>

Gel electrophoresis of nucleic acids is an analytical technique to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules are placed on a gel, where an electric field induces the nucleic acids to migrate toward the positively charged anode. The molecules separate as they travel through the gel based on the each molecule's size and shape. Longer molecules move more slowly because they the gel resists their movement more forcefully than it resists shorter molecules. After some time, the electricity is turned off and the positions of the different molecules are analyzed.

<span class="mw-page-title-main">Two-dimensional gel electrophoresis</span>

Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Coomassie brilliant blue</span> Chemical compound

Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from Coomassie brilliant blue R-250 by the addition of two methyl groups. The name "Coomassie" is a registered trademark of Imperial Chemical Industries.

<span class="mw-page-title-main">Isoelectric focusing</span> Type of electrophoresis

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.

<span class="mw-page-title-main">Zymography</span> Electrophoretic hydrolytic enzyme detection technique

Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. Three types of zymography are used; in gel zymography, in situ zymography and in vivo zymography. For instance, gelatin embedded in a polyacrylamide gel will be digested by active gelatinases run through the gel. After Coomassie staining, areas of degradation are visible as clear bands against a darkly stained background.

In pathology, silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels.

<span class="mw-page-title-main">Molecular-weight size marker</span> Set of standards

A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.

QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point and by continuous elution from a gel column.

<span class="mw-page-title-main">Electrophoretic color marker</span>

An electrophoretic color marker is a chemical used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. The color markers are made up of a mixture of dyes that migrate through the gel matrix alongside the sample of interest. They are typically designed to have different mobilities from the sample components and to generate colored bands that can be used to assess the migration and separation of sample components.

<span class="mw-page-title-main">Affinity electrophoresis</span>

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.

Free-flow electrophoresis (FFE), also known as carrier-free electrophoresis, is a matrix-free, high-voltage electrophoretic separation technique. FFE is an analogous technique to capillary electrophoresis, with a comparable resolution, that can be used for scientific questions, where semi-preparative and preparative amounts of samples are needed. It is used to quantitatively separate samples according to differences in charge or isoelectric point by forming a pH gradient. Because of the versatility of the technique, a wide range of protocols for the separation of samples like rare metal ions, protein isoforms, multiprotein complexes, peptides, organelles, cells, DNA origami, blood serum and nanoparticles exist. The advantage of FFE is the fast and gentle separation of samples dissolved in a liquid solvent without any need of a matrix, like polyacrylamide in gel electrophoresis. This ensures a very high recovery rate since analytes do not adhere to any carrier or matrix structure. Because of its continuous nature and high volume throughput, this technique allows a fast separation of preparative amounts of samples with a very high resolution. Furthermore, the separations can be conducted under native or denaturing conditions.

<span class="mw-page-title-main">Discontinuous electrophoresis</span> Type of laboratory technique

Discontinuous electrophoresis is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.

<span class="mw-page-title-main">SDS-PAGE</span> Biochemical technique

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.

References

  1. ↑ Michov, Budin (2022). Electrophoresis Fundamentals: Essential Theory and Practice. De Gruyter. p. 490. doi:10.1515/9783110761641. ISBN   9783110761641. S2CID   247987700.
  2. ↑ Stringer, R. (2005). "Electrophoresis". Encyclopedia of Analytical Science (2nd ed.). Royal Liverpool University Hospital. p. 360. doi:10.1016/B0-12-369397-7/00120-5. ISBN   978-0-12-369397-6.
  3. ↑ Meredith, S C (1984). "The determination of molecular weight of proteins by gel permeation chromatography in organic solvents". Journal of Biological Chemistry. 259 (19): 11682–11685. doi: 10.1016/s0021-9258(20)71263-9 . ISSN   0021-9258. PMID   6480578.
  4. ↑ Eubel, Holger; Braun, Hans-Peter; Millar, AHarvey (2005). "Blue-native PAGE in plants: a tool in analysis of protein-protein interactions". Plant Methods. 1 (1): 11. doi: 10.1186/1746-4811-1-11 . ISSN   1746-4811. PMC   1308860 . PMID   16287510.
  5. ↑ Schägger, Hermann; von Jagow, Gebhard (1991). "Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form". Analytical Biochemistry. 199 (2): 223–231. doi:10.1016/0003-2697(91)90094-A. PMID   1812789.
  6. ↑ Wittig, Ilka; Braun, Hans-Peter; Schägger, Hermann (2006). "Blue native PAGE". Nat. Protoc. 1 (1): 418–428. doi:10.1038/nprot.2006.62. PMID   17406264. S2CID   19715017.
  7. 1 2 Wittig, IIlka; Schägger, Hermann (2005-10-11). "Advantages and limitations of clear-native PAGE". Proteomics. 5 (17): 4338–4346. doi:10.1002/pmic.200500081. PMID   16220535. S2CID   23396231.
  8. ↑ Gavin, Paul D.; Devenish, Rodney J.; Prescott, Mark (2003). "FRET reveals changes in the F1–stator stalk interaction during activity of F1F0-ATP synthase". Biochim Biophys Acta. 1607 (2–3): 167–79. doi: 10.1016/j.bbabio.2003.09.013 . PMID   14670607.
  9. ↑ Kastenholz, Bernd (2004). "Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE): An Efficient Method for Isolating Cadmium Cofactors in Biological Systems". Analytical Letters. 37 (4): 657–665. doi:10.1081/AL-120029742. ISSN   0003-2719. S2CID   97636537.
  10. ↑ Ornstein, Leonard (December 1964). "Disc Electrophoresis. I. Background and Theory". Annals of the New York Academy of Sciences. 121 (2): 321–349. Bibcode:1964NYASA.121..321O. CiteSeerX   10.1.1.140.7598 . doi:10.1111/j.1749-6632.1964.tb14207.x. PMID   14240533. S2CID   28591995.
  11. ↑ Davis, Baruch J. (December 1964). "Disc Electrophoresis. 2, Method and application to human serum proteins". Annals of the New York Academy of Sciences. 121 (2): 404–427. Bibcode:1964NYASA.121..404D. doi:10.1111/j.1749-6632.1964.tb14213.x. PMID   14240539. S2CID   30512118.
  12. ↑ Schägger, Hermann; von Jagow, Gebhard (1987-11-01). "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa". Analytical Biochemistry. 166 (2): 368–379. doi:10.1016/0003-2697(87)90587-2. ISSN   0003-2697. PMID   2449095.
  13. ↑ Wiltfang, Jens; Arold, Norbert; Neuhoff, Volker (1991). "A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100 000–1000, and their detection with picomolar sensitivity". Electrophoresis. 12 (5): 352–366. doi:10.1002/elps.1150120507. ISSN   0173-0835. PMID   1718736. S2CID   40101706.
  14. ↑ Kohlrausch, Friedr (1897). "Ueber Concentrations-Verschiebungen durch Electrolyse im Inneren von Lösungen und Lösungsgemischen". Annalen der Physik und Chemie. 62 (10): 209–239. Bibcode:1897AnP...298..209K. doi:10.1002/andp.18972981002.
  15. ↑ Westermeier, Reiner (2016-05-02). Electrophoresis in Practice: Guide to Methods and Applications of DNA and Protein Separations, A (5th ed.). Wiley. p. 43. doi:10.1002/9783527695188. ISBN   978-3-527-69518-8.
  16. ↑ Westermeier, Reiner (2016-02-26). Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations (5th ed.). Wiley. doi:10.1002/9783527695188. ISBN   978-3-527-69518-8.
  17. 1 2 3 Sasse, Joachim; Gallagher, Sean R. (2009). "Staining Proteins in Gels". Current Protocols in Molecular Biology. 85 (1): Unit 10.6. doi:10.1002/0471142727.mb1006s85. ISSN   1934-3639. PMID   19170026. S2CID   205153300.
  18. 1 2 Singer, Victoria L.; Haugland, Richard P. (1999). "Fluorescent Imaging of Nucleic Acids and Proteins in Gels". Fluorescent and Luminescent Probes for Biological Activity (2nd ed.). Academic Press. pp. 58–61. doi:10.1016/b978-012447836-7/50006-3. ISBN   978-0-12-447836-7.
  19. ↑ "In-gel Fluorescence". Azure Biosystems. Retrieved 2023-12-02.
  20. ↑ Westermeier, Reiner (2016-05-02). Electrophoresis in Practice: Guide to Methods and Applications of DNA and Protein Separations, A (5th ed.). Wiley. pp. 2, 12, 121. doi:10.1002/9783527695188. ISBN   978-3-527-69518-8.
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