View source for SDS-PAGE

{{short description|Biochemical technique}}
[[File:RBC Membrane Proteins SDS-PAGE gel.jpg|thumb|Proteins of the [[erythrocyte]] membrane separated by SDS-PAGE according to their molecular masses]]
'''SDS-PAGE''' ('''sodium dodecyl sulfate–polyacrylamide gel electrophoresis''') is a [[Discontinuous electrophoresis|discontinuous electrophoretic system]] developed by [[Ulrich K. Laemmli]] which is commonly used as a method to separate [[protein]]s with molecular masses between 5 and 250 [[Kilodalton|kDa]].<ref name="Laemmli1970">{{cite journal|last1=Laemmli|first1=U. K.|title=Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4|journal=Nature|volume=227|issue=5259|year=1970|pages=680–685|issn=0028-0836| doi= 10.1038/227680a0 |pmid= 5432063 |bibcode= 1970Natur.227..680L| s2cid=3105149}}</ref><ref>{{cite news| url= http://www.nzzfolio.ch/www/d80bd71b-b264-4db4-afd0-277884b93470/showarticle/e3c7a889-34c2-407a-813f-22830be78282.aspx |title= Interview with Ulrich Lämmli | language= de| work= Neue Zürcher Zeitung| number= 11| year= 2005| accessdate= March 4, 2012}}</ref> The combined use of [[sodium dodecyl sulfate]] (SDS, also known as sodium lauryl 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.<ref>Neue Züricher Zeitung: [http://www.nzzfolio.ch/www/d80bd71b-b264-4db4-afd0-277884b93470/showarticle/e3c7a889-34c2-407a-813f-22830be78282.aspx Interview with Ulrich Lämmli (in German)]. NZZ Folio, No. 11, 2005. Accessed March 4, 2012.</ref>
== Properties ==
[[File:Protein-SDS interaction.png|thumb|Unfolding of a protein with SDS]]
[[File:Process of Denaturation.svg|thumb|Unfolding of a protein with heat]]
SDS-PAGE is an [[electrophoresis]] method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. The polyacrylamide-gel is typically sandwiched between two glass plates in a [[slab gel]]. Although [[tube gels]] (in glass cylinders) were used historically, they were rapidly made obsolete with the invention of the more convenient slab gels.<ref name="Studier2000">{{cite journal | last=Studier | first=F | title=Slab-gel electrophoresis | journal=Trends in Biochemical Sciences | publisher=Elsevier BV | volume=25 | issue=12 | date=2000-12-01 | issn=0968-0004 | doi=10.1016/s0968-0004(00)01679-0 | pages=588–590| pmid=11116182 }}</ref> In addition, SDS ([[sodium dodecyl sulfate]]) is used. About 1.4 grams of SDS bind to a gram of protein,<ref name="Reynolds">{{cite journal| last1= Reynolds |first1= JA| last2= Tanford| first2= Charles| authorlink2= Charles Tanford | title=Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. | journal=Proc Natl Acad Sci U S A | year= 1970 | volume= 66 | issue= 3 | pages= 1002–7 | pmid= 5269225 | doi= 10.1073/pnas.66.3.1002| pmc=283150 | bibcode=1970PNAS...66.1002R |doi-access= free}}</ref><ref name="Smith1984">{{cite book|last1=Smith|first1=B. J.| title= Proteins |series= Methods in Molecular Biology|volume=1|year=1984|pages=41–56|doi=10.1385/0-89603-062-8:41|pmid=20512673|isbn=0-89603-062-8|chapter=SDS Polyacrylamide Gel Electrophoresis of Proteins}}</ref><ref name="StaikosDondos2009">{{cite journal| last1= Staikos |first1= Georgios |last2= Dondos |first2= Anastasios|title=Study of the sodium dodecyl sulphate–protein complexes: evidence of their wormlike conformation by treating them as random coil polymers|journal=Colloid and Polymer Science|volume=287|issue=8|year=2009|pages=1001–1004|issn=0303-402X| doi=10.1007/s00396-009-2059-3| s2cid= 97367384}}</ref> corresponding to one SDS molecule charges per two [[amino acid]]s.<ref name="Ninfa">{{Cite book|title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology.|vauthors=Ninfa AJ, Ballou DP, Benore M|publisher=John Wiley & Sons, Inc.|year=2010|isbn=978-0-470-08766-4|edition=2nd|location=Hoboken, NJ|oclc=420027217}}</ref> SDS acts as a [[surfactant]], masking the protein's intrinsic charge and conferring them very similar charge-to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of a separating gel. Upon application of a constant electric field, the proteins migrate towards the anode, each with a different speed, depending on their mass. This simple procedure allows precise protein separation by mass.
SDS tends to form spherical [[micelle]]s in aqueous solutions above a certain concentration called the [[critical micellar concentration]] (CMC). Above the critical micellar concentration of 7 to 10 millimolar in solutions, the SDS simultaneously occurs as single molecules ([[monomer]]) and as micelles, below the CMC SDS occurs only as monomers in aqueous solutions. At the critical micellar concentration, a micelle consists of about 62 SDS molecules.<ref name="TurroYekta1978">{{cite journal|last1=Turro|first1=Nicholas J.|last2=Yekta|first2=Ahmad|title=Luminescent probes for detergent solutions. A simple procedure for determination of the mean aggregation number of micelles|journal=Journal of the American Chemical Society|volume=100|issue=18|year=1978|pages=5951–5952|issn=0002-7863|doi=10.1021/ja00486a062}}</ref> However, only SDS monomers bind to proteins via hydrophobic interactions, whereas the SDS micelles are anionic on the outside and do not adsorb any protein.<ref name="Reynolds"/> SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure.<ref>{{Cite book|title=Biochemistry| last1= Berg| first1= Jeremy M. |others=Tymoczko, John L.; Gatto, Gregory J. Jr.; Stryer, Lubert|isbn=9781464126109|edition= Eighth |location=New York|oclc=913469736|date = 2015-04-08}}</ref> In SDS concentrations above 0.1 millimolar, the unfolding of proteins begins,<ref name="Reynolds"/> and above 1 mM, most proteins are denatured.<ref name="Reynolds"/> Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, [[quaternary structure]]s can generally not be determined with SDS. Exceptions are proteins that are stabilised by covalent [[cross-link]]ing (e.g. -S-S- linkages) and the SDS-resistant protein complexes, which are stable even in the presence of SDS (the latter, however, only at room temperature). To denature the SDS-resistant complexes a high activation energy is required, which is achieved by heating. SDS resistance is based on a metastability of the protein fold. Although the native, fully folded, SDS-resistant protein does not have sufficient stability in the presence of SDS, the [[chemical equilibrium]] of denaturation at room temperature occurs slowly. Stable protein complexes are characterised not only by SDS resistance but also by stability against [[protease]]s and an increased [[biological half-life]].<ref name="Manning">{{cite journal|vauthors=Manning M, Colón W | title=Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure. | journal=Biochemistry | year= 2004 | volume= 43 | issue= 35 | pages= 11248–54 | pmid=15366934 | doi=10.1021/bi0491898 | url=https://pubmed.ncbi.nlm.nih.gov/15366934}}</ref>
Alternatively, polyacrylamide gel electrophoresis can also be performed with the cationic surfactants [[Cetrimonium bromide|CTAB]] in a CTAB-PAGE,<ref name="Buxbaum2003">{{cite journal|last1=Buxbaum|first1=Engelbert|title=Cationic electrophoresis and electrotransfer of membrane glycoproteins|journal=Analytical Biochemistry|volume=314|issue=1|year=2003|pages=70–76|issn=0003-2697|doi=10.1016/S0003-2697(02)00639-5|pmid=12633604}}</ref><ref name="AkinShapira1985">{{cite journal|last1=Akin|first1=Dianne T.|last2=Shapira|first2=Raymond|last3=Kinkade|first3=Joseph M.|title=The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis|journal=Analytical Biochemistry|volume=145|issue=1|year=1985|pages=170–176|issn=0003-2697|doi=10.1016/0003-2697(85)90343-4|pmid=4003759}}</ref><ref name="Simpson2010">{{cite journal|last1=Simpson|first1=R. J.|title=CTAB-PAGE|journal=Cold Spring Harbor Protocols|volume=2010|issue=4|year=2010|pages=pdb.prot5412|issn=1559-6095|doi=10.1101/pdb.prot5412|pmid=20360366}}</ref> or [[16-BAC]] in a BAC-PAGE.<ref name="HartingerStenius1996">{{cite journal|last1=Hartinger|first1=Joachim|last2=Stenius|first2=Katinka|last3=Högemann|first3=Dagmar|last4=Jahn|first4=Reinhard| title=16-BAC/SDS–PAGE: A Two-Dimensional Gel Electrophoresis System Suitable for the Separation of Integral Membrane Proteins|journal=Analytical Biochemistry|volume=240|issue=1|year=1996|pages=126–133|issn=0003-2697|doi=10.1006/abio.1996.0339|pmid=8811889|doi-access=free}}</ref>
== Procedure ==
The SDS-PAGE method is composed of gel preparation, sample preparation, electrophoresis, protein staining or [[western blot]]ting and analysis of the generated banding pattern.
=== Gel production ===
[[File:Electrophoresis gel combs 2.jpg|thumb|Sample combs with different numbers of pockets, each prong leaves a pocket in the gel when pulled out]]
[[File:Acrylamidgel.JPG|thumb|Polymerised separating and stacking gel before removing the sample comb (white) between the spacers (black), in the stacking gel are small amounts of bromophenol blue for improved visibility, the separating gel is unstained]]
When using different buffers in the gel (discontinuous gel electrophoresis), the gels are made up to one day prior to electrophoresis, so that the [[diffusion]] does not lead to a mixing of the buffers. The gel is produced by [[free radical polymerization]] in a mold consisting of two sealed glass plates with spacers between the glass plates. In a typical mini-gel setting, the spacers have a thickness of 0.75&nbsp;mm or 1.5&nbsp;mm, which determines the loading capacity of the gel. For pouring the gel solution, the plates are usually clamped in a stand which temporarily seals the otherwise open underside of the glass plates with the two spacers. For the gel solution, acrylamide is mixed as gel-former (usually 4% V/V in the stacking gel and 10-12 % in the separating gel), methylenebisacrylamide as a cross-linker, stacking or separating gel buffer, water and SDS. By adding the catalyst [[TEMED]] and the radical initiator [[ammonium persulfate]] (APS) the polymerisation is started.<ref name="Brunelle">J. L. Brunelle, R. Green: ''One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).'' In: ''Methods in enzymology.'' Volume 541, 2014, p.&nbsp;151–159, {{doi|10.1016/B978-0-12-420119-4.00012-4}}, PMID 24674069.</ref> The solution is then poured between the glass plates without creating bubbles. Depending on the amount of catalyst and radical starter and depending on the temperature, the polymerisation lasts between a quarter of an hour and several hours. The lower gel (separating gel) is poured first and covered with a few drops of a barely water-soluble alcohol (usually buffer-saturated butanol or isopropanol), which eliminates bubbles from the [[Meniscus (liquid)|meniscus]] and protects the gel solution of the [[radical scavenger]] oxygen. After the polymerisation of the separating gel, the alcohol is discarded and the residual alcohol is removed with [[filter paper]]. After addition of APS and TEMED to the stacking gel solution, it is poured on top of the solid separation gel. Afterwards, a suitable sample comb is inserted between the glass plates without creating bubbles. The sample comb is carefully pulled out after polymerisation, leaving pockets for the sample application. For later use of proteins for [[protein sequencing]], the gels are often prepared the day before electrophoresis to reduce reactions of unpolymerised acrylamide with [[cysteine]]s in proteins.
By using a [[gradient mixer]], gradient gels with a gradient of acrylamide (usually from 4 to 12%) can be cast, which have a larger separation range of the molecular masses.<ref name="Margolis">{{cite journal|vauthors=Margolis J, Kenrick KG | title=2-dimensional resolution of plasma proteins by combination of polyacrylamide disc and gradient gel electrophoresis. | journal=Nature | year= 1969 | volume= 221 | issue= 5185 | pages= 1056–7 | pmid=5774398 | doi= 10.1038/2211056a0| url=https://pubmed.ncbi.nlm.nih.gov/5774398| bibcode=1969Natur.221.1056M | s2cid=4197850 }}</ref> Commercial gel systems (so-called ''pre-cast gels'') usually use the buffer substance [[Bis-tris methane]] with a pH value between 6.4 and 7.2 both in the stacking gel and in the separating gel.<ref name="HachmannAmshey2005">{{cite journal|last1=Hachmann|first1=John P.|last2=Amshey|first2=Joseph W.|title=Models of protein modification in Tris–glycine and neutral pH Bis–Tris gels during electrophoresis: Effect of gel pH|journal=Analytical Biochemistry|volume=342|issue=2|year=2005|pages=237–245|issn=0003-2697|doi=10.1016/j.ab.2005.04.015|pmid=15935323}}</ref><ref name="WiltfangArold1991">{{cite journal|last1=Wiltfang|first1=Jens|last2=Arold|first2=Norbert|last3=Neuhoff|first3=Volker|title=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|journal=Electrophoresis|volume=12|issue=5|year=1991|pages=352–366|issn=0173-0835|doi=10.1002/elps.1150120507|pmid=1718736|s2cid=40101706}}</ref> These gels are delivered cast and ready-to-use. Since they use only one buffer ([[continuous gel electrophoresis]]) and have a nearly neutral pH, they can be stored for several weeks. The more neutral pH slows the hydrolysis and thus the decomposition of the polyacrylamide. Furthermore, there are fewer acrylamide-modified cysteines in the proteins.<ref name="HachmannAmshey2005"/> Due to the constant pH in collecting and separating gel there is no stacking effect. Proteins in BisTris gels can not be stained with ruthenium complexes.<ref name="MoebiusDenker2007">{{cite journal|last1=Moebius|first1=Jan|last2=Denker|first2=Katrin|last3=Sickmann|first3=Albert|title=Ruthenium (II) tris-bathophenanthroline disulfonate is well suitable for Tris-Glycine PAGE but not for Bis-Tris gels|journal=Proteomics|volume=7|issue=4|year=2007|pages=524–527|issn=1615-9853|doi=10.1002/pmic.200600642|pmid=17309097|s2cid=25822873}}</ref> This gel system has a comparatively large separation range, which can be varied by using [[MES (buffer)|MES]] or [[MOPS]] in the running buffer.<ref name="HachmannAmshey2005"/>
=== Sample preparation ===
[[File:Disulfide reduction by DTT-2.png|thumb|Disulfide reduction by DTT]]
During sample preparation, the sample buffer, and thus SDS, is added in excess to the proteins, and the sample is then heated to 95&nbsp;°C for five minutes, or alternatively 70&nbsp;°C for ten minutes. Heating disrupts the [[secondary structure|secondary]] and [[tertiary structure]]s of the protein by disrupting [[hydrogen bond]]s and stretching the molecules. Optionally, [[disulfide bridge]]s can be cleaved by reduction. For this purpose, reducing [[thiol]]s such as [[β-mercaptoethanol]] (β-ME, 5% by volume), [[dithiothreitol]] (DTT, 10–100 millimolar),<ref name="Woodland">Breyer Woodland, Aleksandar Necakov, Jens R. Coorssen: ''Optimized Proteome Reduction for Integrative Top–Down Proteomics.'' In: ''Proteomes.'' 2023, Band 11, Nummer 1, S.&nbsp;10 {{doi|10.3390/proteomes11010010}}. PMID 36976889. {{PMC|10059017}}.</ref> [[dithioerythritol]] (DTE, 10 millimolar), [[tris(2-carboxyethyl)phosphine]]<ref name="DOI10.1021/jo00008a014">John A. Burns, James C. Butler, John T. Moran, George M. Whitesides: ''Selective reduction of disulfides by tris(2-carboxyethyl)phosphine.'' In: ''Journal of organic chemistry.'' 1991, Band 56, Nummer 8, S.&nbsp;2648–2650 {{doi|10.1021/jo00008a014}}.</ref> or [[tributylphosphine]]<ref name="Woodland" /> are added to the sample buffer. After cooling to room temperature, each sample is pipetted into its own well in the gel, which was previously immersed in electrophoresis buffer in the electrophoresis apparatus.
In addition to the samples, a [[molecular-weight size marker]] is usually loaded onto the gel. This consists of proteins of known sizes and thereby allows the estimation (with an error of ± 10%) of the sizes of the proteins in the actual samples, which migrate in parallel in different tracks of the gel.<ref name="Rosenberg2006">{{cite book| first= Ian M. |last= Rosenberg|title=Protein Analysis and Purification: Benchtop Techniques |url= https://books.google.com/books?id=gi-UgCF8G6EC&pg=PA103|date=22 December 2006|publisher=Springer Science & Business Media|isbn=978-0-8176-4412-3 |pages= 103–}}</ref> The size marker is often pipetted into the first or last pocket of a gel.
=== Electrophoresis ===
[[File:Gelelektrophoreseapparatur.jpg|thumb|Electrophoresis chamber after a few minutes of electrophoresis. In the first pocket a size marker was applied with bromophenol blue, in the other pockets, the samples were added [[bromocresol green]]]]
[[File:SDSPAGE.jpg|thumb|Electrophoresis chamber after an hour of electrophoresis at 80 Volts. In the first and the last two wells loaded, a commercial protein ladder was applied. The other loaded wells contain protein samples coated in SDS.]]
For separation, the denatured samples are loaded onto a gel of polyacrylamide, which is placed in an electrophoresis buffer with suitable electrolytes. Thereafter, a [[voltage]] (usually around 100 V, 10-20 V per cm gel length) is applied, which causes a migration of negatively charged molecules through the gel in the direction of the positively charged [[anode]]. The gel acts like a sieve. Small proteins migrate relatively easily through the mesh of the gel, while larger proteins are more likely to be retained and thereby migrate more slowly through the gel, thereby allowing proteins to be separated by molecular size. The electrophoresis lasts between half an hour to several hours depending on the voltage and length of gel used.
The fastest-migrating proteins (with a molecular weight of less than 5 kDa) form the buffer front together with the anionic components of the electrophoresis buffer, which also migrate through the gel. The area of the buffer front is made visible by adding the comparatively small, anionic dye [[bromophenol blue]] to the sample buffer. Due to the relatively small molecule size of bromophenol blue, it migrates faster than proteins. By optical control of the migrating colored band, the electrophoresis can be stopped before the dye and also the samples have completely migrated through the gel and leave it.
The most commonly used method is the discontinuous SDS-PAGE.<ref name="Buxbaum 19">E. Buxbaum: ''Cationic Electrophoresis.'' In: ''Methods in molecular biology.'' Volume 1855, 2019, p.&nbsp;115–124, {{doi|10.1007/978-1-4939-8793-1_12}}, PMID 30426413.</ref><ref name="Backman">L. Backman, K. Persson: ''The No-Nonsens SDS-PAGE.'' In: ''Methods in molecular biology.'' Volume 1721, 2018, p.&nbsp;89–94, {{doi|10.1007/978-1-4939-7546-4_8}}, PMID 29423849.</ref> In this method, the proteins migrate first into a collecting gel with neutral pH, in which they are concentrated and then they migrate into a separating gel with basic pH, in which the actual separation takes place. Stacking and separating gels differ by different [[wiktionary:Pore|pore]] size (4-6 % T and 10-20 % T), [[ionic strength]] and [[pH value]]s (pH 6.8 or pH 8.8). The electrolyte most frequently used is an SDS-containing [[Tris]]-[[glycine]]-[[chloride]] [[Buffering agent|buffer]] system. At neutral pH, glycine predominantly forms the [[zwitterionic]] form, at high pH the glycines lose positive charges and become predominantly anionic. In the collection gel, the smaller, negatively charged chloride ions migrate in front of the proteins (as leading ions) and the slightly larger, negatively and partially positively charged glycinate ions migrate behind the proteins (as initial trailing ions), whereas in the comparatively basic separating gel both ions migrate in front of the proteins. The pH gradient between the stacking and separation gel buffers leads to a stacking effect at the border of the stacking gel to the separation gel, since the glycinate partially loses its slowing positive charges as the pH increases and then, as the former trailing ion, overtakes the proteins and becomes a leading ion, which causes the bands of the different proteins (visible after a staining) to become narrower and sharper - the stacking effect. For the separation of smaller proteins and peptides, the TRIS-[[Tricine]] buffer system of Schägger and von Jagow is used due to the higher spread of the proteins in the range of 0.5 to 50 kDa.<ref name="Schäggervon Jagow1987">{{cite journal|last1=Schägger|first1=Hermann|last2=von Jagow|first2=Gebhard|title=Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa|journal=Analytical Biochemistry|volume=166|issue=2|year=1987|pages=368–379|issn=0003-2697|doi=10.1016/0003-2697(87)90587-2|pmid=2449095}}</ref>
=== Gel staining ===
[[File:Coomassie3.jpg|thumb|Coomassie-stained 10% Tris/Tricine gel. In the left lane, a molecular weight size marker was used to estimate the size (from top to bottom: 66, 45, 35, 24, 18 and 9 kDa). In the remaining lanes purified yeast proteins were separated.]]
At the end of the electrophoretic separation, all proteins are sorted by size and can then be analyzed by other methods, e. g. protein staining such as [[Coomassie brilliant blue|Coomassie]] staining (most common and easy to use),<ref>{{ cite journal | last1 = Fazekas de St. Groth | first1 = S. | last2 = Webster | first2 = R. G. | last3 = Datyner | first3 = A. | year = 1963 | title = Two new staining procedures for quantitative estimation of proteins on electrophoretic strips | journal = Biochimica et Biophysica Acta | volume = 71 | pages = 377–391 | pmid = 18421828 | doi = 10.1016/0006-3002(63)91092-8 }}</ref><ref name="Wilson1979">{{cite journal| last= Wilson |first= CM| title=Studies and critique of Amido Black 10B, Coomassie Blue R, and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis. | journal=Anal Biochem | year= 1979 | volume= 96 | issue= 2 | pages= 263–78 | pmid= 89822 | doi= 10.1016/0003-2697(79)90581-5}}</ref> [[silver stain]]ing (highest sensitivity),<ref>{{cite journal | last1 = Merril | first1 = C. R. | last2 = Switzer | first2 = R. C. | last3 = Keuren | first3 = M. L. Van | year = 1979| title = Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. | journal = [[Proc Natl Acad Sci U S A]] | volume = 76 | issue = 9| pages = 4335–4339 | pmid = 92027 | pmc = 411569 | doi = 10.1073/pnas.76.9.4335 | bibcode = 1979PNAS...76.4335M | doi-access = free }}</ref><ref>{{citation|surname1=R. C. Switzer, C. R. Merril, S. Shifrin|periodical=Anal Biochem|title=A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels|volume=98|issue=1|pages=231–237|pmid=94518|date=September 1979|language=de|doi=10.1016/0003-2697(79)90732-2
}}</ref><ref>{{cite journal | last1 = Blum | first1 = H. | last2 = Beier | first2 = H. | last3 = Gross | first3 = H. J. | year = 1987| title = Improved silver staining of plant protein, RNA & DNA in PAA gels. | journal = Electrophoresis | volume = 8 | pages = 93–99 | doi = 10.1002/elps.1150080203 | s2cid = 84471792 }}</ref><ref>{{cite journal | last1 = Rabilloud | first1 = T. | display-authors = etal | year = 1988| title = Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. | journal = Electrophoresis | volume = 9 | issue = 6| pages = 288–291 | pmid = 2466660 | doi = 10.1002/elps.1150090608 | s2cid = 33007991 }}</ref><ref>{{cite journal | last1 = Rabilloud | first1 = T. | year = 1992 | title = A comparison between low background silver diammine and silver nitrate protein stains. | journal = Electrophoresis | volume = 13 | issue = 7| pages = 429–439 | pmid = 1425556 | doi = 10.1002/elps.1150130190 | s2cid = 43084621 }}</ref><ref>{{cite book | last1 = Lelong | first1 = C. | last2 = Chevallet | first2 = M. | last3 = Luche | first3 = S. | last4 = Rabilloud | first4 = T. | title = Two-Dimensional Electrophoresis Protocols | chapter = Silver Staining of Proteins in 2DE Gels | year = 2009 | url = https://hal.archives-ouvertes.fr/hal-00377752/file/Silverstainchapter.pdf| series = Methods Mol Biol. | volume = 519 | pages = 339–350 | pmid = 19381593 | doi = 10.1007/978-1-59745-281-6_21 | isbn = 978-1-58829-937-6 | s2cid = 52820065 }}</ref> [[stains all]] staining, [[Amido black 10B]] staining,<ref name="Wilson1979"/> [[Fast green FCF]] staining,<ref name="Wilson1979"/> fluorescent stains such as [[epicocconone]] stain<ref>{{cite journal | last1 = Moritz | first1 = Christian P. | last2 = Marz | first2 = Sabrina X. | last3 = Reiss | first3 = Ralph | last4 = Schulenborg | first4 = Thomas | last5 = Friauf | first5 = Eckhard | title = Epicocconone staining: a powerful loading control for Western blots | journal = Proteomics | volume =  14| issue = 2–3| pages =  162–8| pmid = 24339236 | doi = 10.1002/pmic.201300089 | date = February 2014 | s2cid = 206368546 }}</ref> and SYPRO orange stain,<ref name="Demchenko">{{cite book| first= Aleksandr Petrovich |last= Demchenko| title= Advanced Fluorescence Reporters in Chemistry and Biology III: Applications in Sensing and Imaging Band 3 von Advanced Fluorescence Reporters in Chemistry and Biology| publisher= Springer |year= 2011| isbn= 978-3-642-18035-4}}</ref> and immunological detection such as the [[Western Blot]].<ref name="GallagherChakavarti2008">{{cite journal |last1= Gallagher |first1=Sean|last2=Chakavarti|first2=Deb|title=Staining Proteins in Gels|journal=Journal of Visualized Experiments |issue= 17 |year= 2008 |issn= 1940-087X|doi=10.3791/760|pmc=3253607|pmid=19066521}}</ref><ref name="Wilson1983">{{cite book| last= Wilson |first= CM|chapter= Staining of proteins on gels: Comparisons of dyes and procedures| title=Enzyme Structure Part I | series=Methods Enzymol | year= 1983 | volume= 91 | pages= 236–47 | pmid=6190068 | doi= 10.1016/s0076-6879(83)91020-0|isbn= 9780121819910| url=https://pubmed.ncbi.nlm.nih.gov/6190068}}</ref> The fluorescent dyes have a comparatively higher linearity between protein quantity and color intensity of about three orders of magnitude above the [[detection limit]] (the quantity of protein that can be estimated by color intensity). When using the fluorescent protein dye [[trichloroethanol]], a subsequent protein staining is omitted if it was added to the gel solution and the gel was irradiated with UV light after electrophoresis.<ref name="Ladner">{{cite journal|vauthors=Ladner CL, Yang J, Turner RJ, Edwards RA | title=Visible fluorescent detection of proteins in polyacrylamide gels without staining. | journal=Anal Biochem | year= 2004 | volume= 326 | issue= 1 | pages= 13–20 | pmid=14769330 | doi=10.1016/j.ab.2003.10.047 | url=https://pubmed.ncbi.nlm.nih.gov/14769330}}</ref><ref name="Gilda">{{cite journal|vauthors=Gilda JE, Gomes AV | title= Stain-Free total protein staining is a superior loading control to β-actin for Western blots. | journal=Anal Biochem | year= 2013 | volume= 440 | issue= 2 | pages= 186–8 | pmid=23747530 | doi=10.1016/j.ab.2013.05.027 | pmc=3809032 }}</ref>
In [[Coomassie brilliant blue|Coomassie]] staining, gel is fixed in a 50% ethanol 10% glacial acetic acid solution for 1 hr. Then the solution is changed for fresh one and after 1 to 12 hrs gel is changed to a staining solution (50% methanol, 10% glacial acetic acid, 0.1% coomassie brilliant blue) followed by destaining changing several times a destaining solution of 40% methanol, 10% glacial acetic acid.
=== Analysis ===
Protein staining in the gel creates a documentable banding pattern of the various proteins.
* [[Glycoprotein]]s have differential levels of [[glycosylations]] and adsorb SDS more unevenly at the glycosylations, resulting in broader and blurred bands.<ref name="Cryo">{{cite book| title=Cryo-EM Part A: Sample Preparation and Data Collection|url=https://books.google.com/books?id=a1mPepNTgxcC&pg=PA28|date=30 September 2010|publisher=Academic Press| isbn=978-0-08-095695-4|pages=28}}</ref>
* [[Membrane protein]]s, because of their [[transmembrane domain]], are often composed of the more hydrophobic amino acids, have lower [[solubility]] in aqueous solutions, tend to bind [[lipid]]s, and tend to precipitate in aqueous solutions due to [[hydrophobic effect]]s when sufficient amounts of detergent are not present. This precipitation manifests itself for membrane proteins in a SDS-PAGE in "tailing" above the band of the transmembrane protein. In this case, more SDS can be used (by using more or more concentrated sample buffer) and the amount of protein in the sample application can be reduced.
* An overloading of the gel with a soluble protein creates a semicircular band of this protein (e. g. in the marker lane of the image at 66 kDa), allowing other proteins with similar molecular weights to be covered.
* A low contrast (as in the marker lane of the image) between bands within a lane indicates either the presence of many proteins (low purity) or, if using purified proteins and a low contrast occurs only below one band, it indicates a proteolytic degradation of the protein, which first causes degradation bands, and after further degradation produces a homogeneous color ("smear") below a band.<ref name="BurgessDeutscher2009">{{cite book| first1= Richard R. | last1= Burgess |first2= Murray P. |last2= Deutscher|title=Guide to Protein Purification|url=https://books.google.com/books?id=f6Lp4yna4hoC&pg=PA184|date=3 November 2009|publisher=Academic Press|isbn=978-0-08-092317-8|pages=184–}}</ref>
The documentation of the banding pattern is usually done by photographing or scanning. For a subsequent recovery of the molecules in individual bands, a [[gel extraction]] can be performed.
=== Archiving ===
[[File:Gel_Blue_Coomassie.jpg|thumb|Two SDS gels after completed separation of the samples and staining in a drying frame]]
After protein staining and documentation of the banding pattern, the polyacrylamide gel can be dried for archival storage.<ref name="Stamova">S. Stamova, I. Michalk, H. Bartsch, M. Bachmann: ''Gel drying methods.'' In: ''Methods in molecular biology.'' Volume 869, 2012, p.&nbsp;433–436, {{doi|10.1007/978-1-61779-821-4_36}}, PMID 22585507.</ref> Proteins can be extracted from it at a later date. The gel is either placed in a drying frame (with or without the use of heat) or in a vacuum dryer. The drying frame consists of two parts, one of which serves as a base for a wet [[cellophane]] film to which the gel and a one percent [[glycerol]] solution are added. Then a second wet cellophane film is applied bubble-free, the second frame part is put on top and the frame is sealed with clips. The removal of the air bubbles avoids a fragmentation of the gel during drying. The water evaporates through the cellophane film. In contrast to the drying frame, a vacuum dryer generates a vacuum and heats the gel to about 50&nbsp;°C.
== Molecular mass determination ==
[[File:LogM over Rf.png|thumb|The proteins of the size marker (black X) show an approximately straight line in the representation of log M over Rf. The molecular weight of the unknown protein (red X) can be determined on the y-axis.]]
For a more accurate determination of the molecular weight, the relative migration distances of the individual protein bands are measured in the separating gel.<ref name="BonnerHargreaves2011">{{cite book| first1= Philip L. R.| last1= Bonner| first2= Alan J. |last2= Hargreaves| title= Basic Bioscience Laboratory Techniques: A Pocket Guide|url=https://books.google.com/books?id=Pavxt_jKi80C&pg=PT140|date=24 August 2011|publisher=John Wiley & Sons| isbn=978-1-119-95644-0|pages=140–}}</ref><ref name="Holtzhauer2006">{{cite book| first= Martin |last= Holtzhauer|title=Basic Methods for the Biochemical Lab| url= https://books.google.com/books?id=lymxOH6cGCoC&pg=PA243|date=13 September 2006|publisher=Springer Science & Business Media |isbn= 978-3-540-32786-8| pages=243–}}</ref> The measurements are usually performed in triplicate for increased accuracy. The relative mobility (called Rf value or Rm value) is defined as the distance migrated by the protein band divided by the distance migrated by the buffer front. The distances are each measured from the beginning of the separation gel. The migration of the buffer front roughly corresponds to the migration of the dye contained in the sample buffer. The Rf's of the size marker are plotted semi-logarithmically against their known molecular weights. By comparison with the linear part of the generated graph or by a regression analysis, the molecular weight of an unknown protein can be determined by its relative mobility.<ref name="BonnerHargreaves2011"/><ref>{{cite web |last1=Caprette |first1=David R |title=Measuring mobility of protein bands |url=https://www.ruf.rice.edu/~bioslabs/studies/sds-page/rf.html |website=www.ruf.rice.edu |date=January 5, 2007}}</ref>
Bands of proteins with glycosylations can be blurred,<ref name="Cryo"/> as glycosylation is often heterogenous.<ref>{{cite web |title=Beginners Guide To Glycosylation Of Proteins |url=https://peakproteins.com/beginners-guide-to-glycosylation-of-proteins/ |website=Peak Proteins |date=25 February 2021}}</ref> Proteins with many basic amino acids (e. g. [[histone]]s)<ref name="VenrooijMaini2012">{{cite book| first1= W. J. |last1= van Venrooij| first2= Ravinder N. |last2= Maini |title= Manual of Biological Markers of Disease|url=https://books.google.com/books?id=0ngrBgAAQBAJ&pg=PT50|date=6 December 2012|publisher=Springer Science & Business Media|isbn=978-94-011-1670-1|pages=50–}}</ref> can lead to an overestimation of the molecular weight or even not migrate into the gel at all, because they move slower in the electrophoresis due to the positive charges or even to the opposite direction. On the other hand, many acidic amino acids can lead to accelerated migration of a protein and an underestimation of its molecular mass.<ref name="GuanZhu2015">{{cite journal| last1= Guan| first1= Yihong| last2= Zhu|first2=Qinfang|last3=Huang|first3=Delai|last4=Zhao|first4=Shuyi|last5=Jan Lo| first5= Li| last6= Peng| first6= Jinrong| display-authors= 3| title=An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide |journal= Scientific Reports|volume=5|issue=1|pages=13370|year=2015|issn=2045-2322 |doi= 10.1038/srep13370| pmc=4550835| pmid=26311515 |bibcode= 2015NatSR...513370G}}</ref>
== Applications ==
The SDS-PAGE in combination with a protein stain is widely used in biochemistry for the quick and exact separation and subsequent analysis of proteins. It has comparatively low instrument and reagent costs and is an easy-to-use method. Because of its low [[scalability]], it is mostly used for analytical purposes and less for preparative purposes, especially when larger amounts of a protein are to be isolated.
Additionally, SDS-PAGE is used in combination with the [[western blot]] for the determination of the presence of a specific protein in a mixture of proteins - or for the analysis of [[post-translational modification]]s.<ref name="Mahmood">{{cite journal | vauthors = Mahmood T, Yang PC | title = Western blot: technique, theory, and trouble shooting | journal = North American Journal of Medical Sciences | volume = 4 | issue = 9 | pages = 429–434 | date = September 2012 | pmid = 23050259 | pmc = 3456489 | doi = 10.4103/1947-2714.100998 | doi-broken-date = 1 November 2024 | doi-access = free }}</ref><ref name="Begum">{{cite journal | vauthors = Begum H, Murugesan P, Tangutur AD | title = Western blotting: a powerful staple in scientific and biomedical research | journal = BioTechniques | volume = 73 | issue = 1 | pages = 58–69 | date = June 2022 | pmid = 35775367 | doi = 10.2144/btn-2022-0003 | s2cid = 250175915 | doi-access = free }}</ref> Post-translational modifications of proteins can lead to a different relative mobility (i.e. a ''band shift'') or to a change in the binding of a detection antibody used in the western blot (i.e. a band disappears or appears).
In [[mass spectrometry]] of proteins, SDS-PAGE is a widely used method for sample preparation prior to spectrometry, mostly using [[in-gel digestion]]. In regards to determining the molecular mass of a protein, the SDS-PAGE is a bit more exact than an analytical [[ultracentrifugation]], but less exact than a [[mass spectrometry]] or - ignoring post-translational modifications - a calculation of the protein molecular mass from the [[DNA sequence]].
In medical diagnostics, SDS-PAGE is used as part of the [[HIV test]] and to evaluate [[proteinuria]]. In the HIV test, HIV proteins are separated by SDS-PAGE and subsequently detected by Western Blot with HIV-specific [[antibodies]] of the patient, if they are present in his [[blood serum]]. SDS-PAGE for proteinuria evaluates the levels of various [[serum protein]]s in the urine, e.g. [[Albumin]], [[Alpha-2-macroglobulin]] and [[IgG]].
== Variants ==
SDS-PAGE is the most widely used method for gel electrophoretic separation of proteins. [[Two-dimensional gel electrophoresis]] sequentially combines [[isoelectric focusing]] or BAC-PAGE with a SDS-PAGE.<ref>{{cite journal |author=O'Farrell, PH |year=1975 |title=High resolution two-dimensional electrophoresis of proteins |journal=J. Biol. Chem. |volume=250 |pages=4007–21 |pmc=2874754 |pmid=236308 |issue=10|doi=10.1016/S0021-9258(19)41496-8 |doi-access=free }}</ref><ref>{{Cite journal |pmid=1093965|year=1975|last1=Klose|first1=J|title=Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals|journal=Humangenetik|volume=26|issue=3|pages=231–43|doi=10.1007/bf00281458|s2cid=30981877 |url=https://link.springer.com/article/10.1007/BF00281458|url-access=subscription}}</ref> [[Native PAGE]] is used if native protein folding is to be maintained. For separation of membrane proteins, BAC-PAGE or CTAB-PAGE may be used as an alternative to SDS-PAGE. For electrophoretic separation of larger protein complexes, [[agarose gel electrophoresis]] can be used, e.g. the [[SDD-AGE]]. Some [[enzyme]]s can be detected via their [[enzyme activity]] by [[zymography]].<ref name="vandoorenetal">{{cite journal | vauthors = Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G | year = 2013 | title = Zymography methods for visualizing hydrolytic enzymes | journal = Nat Methods | volume = 10 | issue = 3| pages = 211–220 | doi = 10.1038/nmeth.2371 | pmid = 23443633 | s2cid = 5314901 }}</ref>
== Alternatives ==
While being one of the more precise and low-cost protein separation and analysis methods, the SDS-PAGE denatures proteins. Where non-denaturing conditions are necessary, proteins are separated by a native PAGE or different [[chromatography|chromatographic]] methods with subsequent [[Photometry (optics)|photometric]] [[Quantification (science)|quantification]], for example [[affinity chromatography]] (or even [[tandem affinity purification]]), [[size exclusion chromatography]], [[ion exchange chromatography]].<ref name="Janson2012">{{cite book| first= Jan-Christer |last= Janson|title=Protein Purification: Principles, High Resolution Methods, and Applications|date=3 January 2012|publisher=John Wiley & Sons|isbn=978-1-118-00219-3}}</ref> Proteins can also be separated by size in a [[tangential flow filtration]]<ref name="Desai2000">{{cite book| first= Mohamed A.| last= Desai |title= Downstream Processing of Proteins: Methods and Protocols|year=2000|publisher=Springer Science & Business Media|isbn=978-1-59259-027-8|pages=35}}</ref> or an [[ultrafiltration]].<ref name="Raja2003">{{cite book| first= Ghosh |last= Raja|title=Protein Bioseparation Using Ultrafiltration: Theory, Applications And New Developments|date=11 June 2003|publisher=World Scientific|isbn=978-1-78326-126-0|pages=142}}</ref> Single proteins can be isolated from a mixture by affinity chromatography or by a [[pull-down assay]]. Some historically early and cost effective but crude separation methods usually based upon a series of [[Extraction (chemistry)|extractions]] and [[precipitation]]s using [[kosmotropic]] molecules, for example the [[ammonium sulfate precipitation]] and the [[polyethyleneglycol]] precipitation.
== History ==
In 1948, [[Arne Tiselius]] was awarded the [[Nobel Prize in Chemistry]] for the discovery of the principle of electrophoresis as the migration of charged and dissolved atoms or molecules in an electric field.<ref name="Pederson2007">{{cite journal|last1=Pederson|first1=T.|title=Turning a PAGE: the overnight sensation of SDS-polycrylamide gel electrophoresis|journal=The FASEB Journal|volume=22|issue=4|year=2007|pages=949–953|issn=0892-6638| doi= 10.1096/fj.08-0402ufm|doi-access=free |pmid=18378803|s2cid=33466516}}</ref> The use of a solid matrix (initially paper discs) in a [[zone electrophoresis]] improved the separation. The discontinuous electrophoresis of 1964 by L. Ornstein and B. J. Davis made it possible to improve the separation by the stacking effect.<ref>{{cite journal | last1 = Ornstein | first1 = L. | last2 = Davis | first2 = B. J. | year = 1964 | title = Disc Electrophoresis –1. Background and Theory | journal = Ann NY Acad Sci | volume = 121 | issue = 2| pages = 321–349 | doi = 10.1111/j.1749-6632.1964.tb14207.x | bibcode = 1964NYASA.121..321O | pmid = 14240533 | s2cid = 28591995 }}</ref> The use of cross-linked polyacrylamide hydrogels, in contrast to the previously used paper discs or starch gels, provided a higher stability of the gel and no microbial decomposition. The denaturing effect of SDS in continuous polyacrylamide gels and the consequent improvement in resolution was first described in 1965 by [[David F. Summers]] in the working group of [[James E. Darnell]] to separate poliovirus proteins.<ref name="Summers">{{cite journal|vauthors=Summers DF, Maizel JV, Darnell JE | title=Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. | journal=Proc Natl Acad Sci U S A | year= 1965 | volume= 54 | issue= 2 | pages= 505–13 | pmid= 4285933 | doi= 10.1073/pnas.54.2.505| pmc=219696 | bibcode=1965PNAS...54..505S | doi-access=free }}</ref> The current variant of the SDS-PAGE was described in 1970 by Ulrich K. Laemmli and initially used to characterise the proteins in the head of [[bacteriophage T4]].<ref name="Laemmli1970"/>
== References ==
{{Reflist}}
== External links ==
* [http://openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE,_the_very_best Protocol for BisTris SDS-PAGE] at OpenWetWare.org
[[Category:Electrophoresis]]