AppliChem offers a broad range of products for electrophoretic separation of proteins and nucleic acids. Please select the type of electrophoresis you would like to read more about.
1. Agarose gel electrophoresis, agaroses, running and loading buffers, nucleic acid dyes, DNA size standards.
2. Polyacrylamide gel electrophoresis, acrylamide mixes, buffers for stacking and resolving gels, APS, TEMED, protein size standards, protein dyes.
3. The history of electrophoresis, past and present of one of the most important techniques in life sciences. Interesting details for everyone who wants to know more about the development of his/her everyday methods.
1. Agarose gel electrophoresis
Agarose gel electrophoresis allows separation of DNA or RNA fragments according to their size. In an electric field, the negatively charged nucleic acids migrate to the positively charged cathode. The agarose polymer functions as a molecular sieve: the agarose gel matrix consists of a network with pores of 100-300 nm2, which allow small fragments to rapidly pass through the matrix while larger DNA molecules migrate more slowly.
What is needed? Agarose gels consist of agarose (generally 1-2%), water and buffer substances (TAE or TBE). The buffer which is used for resolving the agarose is also employed as a running buffer during electrophoresis. Prior to its filling into the gel pocket, the DNA sample is mixed with a loading buffer. To visualize the resulting DNA bands, the DNA sample or the gel (prior or after electrophoresis) is stained with a suitable DNA dye, e.g. ethidium bromide or DNA-Dye NonTox. DNA size standards enable an easy evaluation of the fragment sizes.
By choosing a suitable agarose with optimal physical and chemical properties and variation of buffer system and agarose concentration, a very wide range of methods can be covered and huge differences in the separation properties are obtained. Agarose gels provide clear separation pattern of nucleic acids with >50 bp, up to 20-25,000 bp. To find the best agarose for your application, please use our agarose survey.
Larger molecules are still separated by agarose gel electrophoresis, but an adjustments of the electrophoresis protocol is required. For more information on resolution and separation capacity, see Chapter 3 of the brochure "Agarose-Gel-Elektrophorese" (only available in German!).
Agarose is the main component of agar and is isolated from the cell walls of selected red algae (Rhodophyceae). In order to obtain pure agarose with the desired electrophoretic properties, agaropectin (a cell wall "glue" build of branched, often modified and charged polysaccharides) is removed from the agar. The remaining agarose is then chemically modified to yield linear, uncharged molecule that do not interact with proteins and nucleic acids. This characteristic is reflected in low Electroendoosmosis (EEO) values. The EEO value represents the electrophoretic mobility and is a measure for the separating power provided by the agarose matrix. Other important parameters for selection of an appropriate agarose grade are gel point (temperature at which the gel becomes solid), melting point (the temperature at which the gel liquefies) and gel strength. The latter can be seen as an indicator for the gel stability. A high gel strength (expressed as g/cm2) refers to a stable gel which is easy to handle and, however, more suitable for analytical gels and less for preparative applications.
In general, analytical agarose gels are used to visualize differences in the migration pattern and to determine the size of DNA fragments. In contrast, preparative gels are used for selective isolation of a particular fragment. For gel extraction of DNA fragments, please use our DNA Isolation Spin-Kit Agarose or DNA Isolation Spin-Kit Agarose Low Melt AX.
|Prod. No.||Description||EEO||Gel point||Gel strenth; 1.5 % [g/cm2]||(main) Application|
|A8963||Agarose Basic*||36 ± 1.5°C||2200||Economic alternative for Agarose low EEO; standard product for every day applications.|
|A2114||Agarose low EEO* (Agarose Standard)||0.09 - 0.13||36 ± 1.5°C||≥ 2500||Analytic and mainly preparative electrophoresis of nucleic acids, most suitable for fragments ≥1000 bp; blotting; DNA typing.|
|A2116||Agarose medium EEO*||0.16 - 0.19||36 ± 1.5°C||≥ 2000||Analytic nucleic acid separation (fragments ≥1000 bp); Immunoelectrophoresis.|
|A2115||Agarose high EEO*||0.23 - 0.26||36 ± 1.5°C||≥ 1000||(Counter-) Immunoelectrophoresis; separation of serum proteins.|
|A2119||Agarose Low Melt 3||≤ 0.12||24 - 28°C||≥ 550||Preparative electrophoresis of DNA, RNA and proteins as well as in-gel applications. For DNA extraction from low melt agarose gels we recommend DNA Isolation Spin-Kit Agarose Low Melt AX.|
|A3762||Agarose Low Melt Large DNA Grade||≤ 0.12||24 - 28°C||≥ 250 (1% gel)||Analytic and preparative DNA gels with fragments ≥1000 bp; suitable for enzymatic manipulations in the melted gel.|
|A2121||Agarose IMG||≤ 0.12||≤ 35.5°C||≥ 500||Intermediate melting point of ≤70°C (1.5%); analytic DNA gels (fragments ≤1000 bp) with high resolution; blotting.|
|A1091||Agarose MP*||≤ 0.12||≤ 36°C||≥ 3500||Multi-purpose agarose; very good analytic separation (100 bp to 50 kb!); blotting; DNA typing; PFGE.|
*Standard Melting point: 88 ± 1.5°C.
The Low Melt agaroses melt at 65°C, Agarose IMG at ≤80°C.
Electrophoresis buffers and DNA dyes
To prepare the agarose gel, the required amount of agarose is added to a TAE or TBE buffer solution and dissolved by heating (commonly by using a micro-wave). The solubility of agaroses differs. Some dissolve better, some worse (especially when used at higher concentrations). Particularly Low Melt agaroses often have to be autoclaved to dissolve completely. It is important to control the pH; a pH value below 5.5 makes the agarose polymer hydrolyze. As a result, the agarose solution does not solidify! The agarose solution needs to be buffered - pure water (demineralized water is often acidic) cannot be used.In addition to guaranteeing a stable pH, the electrophoresis buffer is required for charge transport. Furthermore, the so-called running buffer (commonly the same buffer in which the agarose is dissolved) that surrounds the gel during electrophoresis is important for cooling the system.
The most commonly used buffer for agarose gel electrophoresis is TAE (Tris-Acetate EDTA) buffer. Originally, it was developed for polyacrylamide gels with a slightly different composition (40 mM Tris; 20 mM NaOAc; 2 mM Na-EDTA; pH 7.8 at +5°C with acetic acid). Today, this buffer is used in a modifed form (40 mM Tris acetate; 1 mM Na-EDTA; ~pH 8.5). TAE has a lower buffer capacity than TBE, but the separation of "supercoiled" DNA is better in TAE then in TBE, and double-stranded linear DNA migrates 10 % faster with the same resolution through TAE-containing agarose gels. Furthermore, TAE buffer does not interact with agarose which is important especially for preparative agarose gel electrophoresis (high yield of nucleic acids).
TBE (Tris-Borate-EDTA) buffer offers a higher buffer capacity than TAE and is more suitable for the separation of small DNA molecules. Due to its interaction with the DNA, TBE is not recommended for preparative gel electrophoresis. A similar alternative to TBE is TPE (Tris-Phosphate-EDTA).
For gels with glyoxal/DMSO-treated RNA and for denaturating formaldehyde-containing agarose gels, MOPS-Acetate-EDTA or BPTE (PIPES, Bis-Tris, EDTA) buffer is commonly employed. Other buffer systems used in agarose gel electrophoresis are TAFE (transverse alternating field electrophoresis buffer, a variant of TAE), GTBE (glycin containing TBE) and TTE (Tris-Taurine-EDTA). More information about different buffer systems including applications, requirements, selection criteria and benefits are described on our website about Biological Buffers.Prior to its loading onto the gel, the DNA/RNA sample is mixed with a loading buffer. This buffer functions as nucleic acid stabilizer, it simplifies the bed down of the sample into the gel slot (facilitated by glycerol or Ficoll® 400, a sucrose-epichlorohydrin copolymer) and it enables the visualization of the running front (tracking dyes).
For staining of the electrophoretically separated DNA mainly ethidium bromide is used. The big advantage of the fluorescent DNA-intercalating dye is its outstanding sensitivity (the detection limit is 1-5 ng DNA per band) and its low price. Unfortunately, ethidium bromide is very toxic and mutagenic. To simplify the handling of this product and to minimize its risks, AppliChem offers a ready-to-use diluted ethidium bromide solution in a handy "dropper bottle".When dealing with ethidium bromide, also the decontamination of the buffer and staining solutions must be considered. For this purpose, AppliChem offers Decontamination bags: tear-resistant charcoal bags for removal of DNA dyes such as ethidium bromide, SYBR Green® or propidium iodide from aqueous solutions. One bag is sufficient for the decontamination of about 500 ml of ethidium bromide solution at concentrations <1 μg/ml.
Alternatives to ethidium bromide are methylene blue (visualization in visible light; the detection limit is 10-200 ng DNA per band) and DNA-Dye Nontox. This fluorescent DNA dye convinces by its high sensitivity (comparable to ethidium bromide) with significantly reduced toxicity and a lack of mutagenicity. For detection, UV or blue light is used.
Buffers and dyes for agarose gel electrophoresis
|A4227||TAE buffer (10X)||Tris-Acetate-EDTA buffer, 10X concentrated aqueous solution|
|A4686||TAE buffer (50X)||Tris-Acetate-EDTA buffer, 50X concentrated aqueous solution|
|A4228||TBE buffer (5X)||Tris-Borate-EDTA buffer, 5X concentrated aqueous solution|
|A3945||TBE buffer (10X)||Tris-Borate-EDTA buffer, 10X concentrated aqueous solution|
|DNA loading buffers (6X)
|A3144||Loading buffer DNA I||For agarose and acrylamide gel electrophoresis; contains bromophenol blue and Ficoll® 400|
|A2571||Loading buffer DNA II||For agarose and acrylamide gel electrophoresis; contains bromophenol blue, xylene cyanol FF and Ficoll® 400|
|A6307||Loading buffer DNA IIb||For agarose and acrylamide gel electrophoresis; contains bromophenol blue, xylene cyanol FF and glycerol|
|A3147||Loading buffer DNA III (alkaline)||For alkaline agarose gel electrophoresis; contains bromocresol green, xylene cyanol, EDTA, NaOH and Ficoll® 400|
|A3481||Loading buffer DNA IV||For agarose gel electrophoresis; contains bromocresol green, xylene cyanol, EDTA, SDS and Ficoll® 400|
|A3476||Loading buffer DNA VIII (for Glyoxal/DMSO - RNA gels)||
7.5X loading buffer for agarose gels for RNA separation; DEPC treated; contains bromophenol blue, xylene cyanol FF, sodium phosphate (pH 7.0) and glycerol.
The RNA probe is denatured in glyoxal/DMSO sodium phosphate buffer and separated on agarose gels.
|DNA dyes for agarose gel electrophoresis
|A1152||Ethidium bromide solution 1%||Highly sensitive fluorescent DNA dye, intercalating; mutagenic.|
|A2273||Ethidium bromid solution 0.07% "dropper bottle"||Enables an easy application; one drop covers the staining of a standard agarose gel (50 ml). For decontamination we recommend A9676, Decontamination-Bags.|
|A9555||DNA-Dye NonTox||Highly sensitive fluorescent DNA dye; non-mutagenic Ethidium bromide substitute; provided ready-to-use in 6X loading buffer|
|A5595||DNA-Dye Methylenblau||Non-toxic DNA dye, 200X concentrated|
DNA size markers
To evaluate the size of the analyzed DNA fragments (e.g. control digest or a PCR product) a size standard employing DNA fragment of defined length in the desired size range is used. AppliChem offers a wide variety of DNA markers (irregular band pattern based on DNA fragments obtained by digestion of specific plasmids) and ladders (DNA ladders, i.e. regular band pattern) for agarose or polyacrylamide gel electrophoresis.
A more detailed description of all AppliChem size standards including handling and storage can be found in our brochure "Gel Electrophoresis Size Marker".
DNA size marker for agarose gel electrophoresis
|Prod. No.||Description||number of bands||size range [bp]|
|A5229||DNA Marker pBR322 - Hae III||22||8 - 587|
|A4406||DNA Marker pBR322 - Hae III (lyophilised)||22||8 - 587|
|A5235||DNA Marker pUC19 - Msp I||12||26 - 501|
|A3996||DNA Marker pUC19 - Msp I (lyophilised)||12||26 - 501|
|A8368||DNA Ladder 50 bp||10||50 - 700|
|A5191||DNA Ladder 100 bp||10||100 - 1000|
|A3470||DNA Ladder 100 bp (lyophilised)||11||100 - 1000|
|A3302||DNA Ladder 100 bp equalized (lyophilised)||11||100 - 1000|
|A5216||DNA Ladder 100 bp plus||11||100 - 1500|
|A6927||DNA Marker pBR328 - Mix (lyophilised)||12||154 - 2176|
|A7215||DNA Marker quick-run (lyophilised)||5||500 - 2500|
|A3660||DNA Ladder Mix 100 - 5000 (lyophilised)||17||100 - 5000|
|A7222||DNA Marker quick-run extended (lyophilised)||9||100 - 6000|
|A5220||DNA Marker Phage Lambda - Bst II||14||117 - 8454|
|A4412||DNA Marker Phage Lambda - Bst II (lyophilised)||14||117 - 8454|
|A3982||DNA Ladder 250 bp (lyophilised)||16||250 - 8000|
|A8640||DNA Ladder 250 bp plus (lyophilised)||15||250 - 12.000|
|A5207||DNA Ladder 1 kb||13||250 - 10.000|
|A2667||DNA Ladder 1 kb (lyophilised)||11||500 - 10.000|
|A5194||DNA Marker Phage Lambda - Sty I||11||74 - 19.329|
|A6430||DNA Ladder 1 kb konkatamer (lyophilised)||~ 25||1000 - ca. 25.000|
|A5223||DNA Marker Phage Lambda - Hind III||8||125 - 23.130|
|A5589||DNA Marker Phage Lambda - Hind III (lyophilised)||8||125 - 23.130|
AppliChem's lyophilised DNA markers meet the highest quality requirements: the DNA digest is not simply resuspended in loading buffer, but phenol extracted, desalted and freeze-dried during production. The DNA starting material originates from special nuclease-poor host bacteria. In addition to their excellent quality our lyophilised DNA markers are characterized by virtually unlimited shelf life and stability even at room temperature - in contrast to ready-to-use DNA markers. Another advantage of the lyophilised form is that the user himself determines the final DNA concentration and therefore is able to optimize the applied volume of the marker (depending on the slot size). Each lyophilised marker is provided together with a loading buffer.Example 1: The desired marker volume per slot is 2 µl, the amount of DNA in the sample is meant to be 1 µg.
1 µg/2 µl = 0.5 µg/µl and
50 µg/0.5 µg/µl = 100; that means 50 µg of the lyophilized marker has to be dissolved in 100 µl of loading buffer.Example 2: The desired marker volume per slot is 10 µl, the amount of DNA is meant to be 0.8 µg.
0,8 µg/10 µl = 0.08 µg/µl and
50 µg/0.08 µg/µl = 625; that means 50 µg of the lyophilized marker has to be dissolved in 625 µl of loading buffer.For combination with the non-toxic fluorescent dye DNA-Dye Nontox the lyophilised markers may be dissolved in TAE buffer instead of loading buffer. DNA-Dye Nontox (already in 6x loading buffer) is added immediately before application. The size spectrum of our lyophilised DNA marker covers all size ranges of the DNA electrophoresis. AppliChem offers products suitable for low (30-700 bp) to high (up to 23,000 bp) size ranges.
2. Polyacrylamide gel electrophoresis, PAGE
Polyacrylamide gel electrophoresis is typically used for separation of proteins, but also DNA (especially to visualize differences between small fragments) can be separated by PAGE. The matrix consists of acrylamide-strands cross-linked wit N,N-methylenebisacrylamide. AppliChem offers reagents and ready mixes for SDS-PAGE, native PAGE, denaturing and non-denaturing DNA-PAGE as well as for sequencing gels. Please choose one of the following product categories:
The separation capacity of a polyacrylamide gel is determined by the mixing ratio of acrylamide to bisacrylamide. The lower the ratio of these two components in the mixture, the higher the degree of crosslinking. This means that a 6 % gel prepared from a stock solution of a mixing ratio of 29 : 1, has a higher degree of crosslinking than a 6 % gel prepared from a stock solution of a mixing ratio of 37.5 : 1.
For most applications an acrylamide : bisacrylamide ratio of 29 : 1 or 37.5 : 1 is used (for electrophoretic separation of nucleic acids or proteins). The mixing ratio of 19: 1 is the solution of choice for DNA sequencing. The preparation is simplified by using 30% or 40% aqueous acrylamide stock solutions with the desired ratio.Acrylamide solutions are often termed as "gas-stabilized", offering increased shelf-life. The "gas" simply is oxygen, which is meant to avoid a spontaneous polymerization of acrylamide solutions. Since the polymerization initiators APS and TEMED are added in excess, however, "degassing" (partly done in Laboratory prior to the polymerization initiation) is actually unnecassary.
The unwanted spontaneous polymerization is started by radicals which typically originate from acrylic acid. Poorer qualities often contain detectable traces of acrylic acid and solutions based on these grades bear a high risk to polymerize spontaneously. In contrast, grades recrystallized four times ("4K"), however, are free of acrylic acid. For our Molecular biology grade only 4K acrylamide especially tested for the absence of DNases, RNases and proteases, is employed!
At low temperatures of 2-8°C, the oxygen exchange is reduced within the acrylamide solution and spontaneous polymerization is facilitated. Therefore, we recommend storage at ambient temperature. The following table lists the most common acrylamide mixes; more acrylamide solutions are available in the shop or on request.
Acrylamide mixes (in water) for PAGE; different grades
|Bezeichnung||"Molecular biology grade"||"4K"|
|Acrylamide solution (30%) Mix 19 : 1||A3857||A0967|
|Acrylamide solution (30%) Mix 29 : 1||A4983||A0951|
|Acrylamide solution (30%) Mix 37.5 : 1||A3626||A1672|
|Acrylamide solution (40%) Mix 37.5 : 1||A4989||A1577|
|Acrylamide solution (63.9 %) Mix 158.75 : 1 for TAU gels||-||A3756|
Usage of acrylamide mixes:
The acrylamide stock solutions are diluted in order to obtain the desired monomer concentration. The total volume of the required amount of gel solution (for example, 100 ml) is divided by the content of the solution (e.g. 30 %) and multiplied with the desired final concentration (for example 6 % acrylamide) to obtain the required volume of the stock solution:
(100 ml / 30 %) * 6 % = 20 ml
To prepare 100 ml of a 6 % acrylamide gel, 20 ml of the 30 % acrylamide stock solution is added into the gel-mixture.Of course, you can request our acrylamide mixes in powder form as well. But caution, the acrylamide monomer is a strong accumulating neurotoxin! Therefore, gloves and a face mask should be worn when handling crystalline acrylamide.
Buffer solutions, SDS, APS, TEMED
The most commonly used electrophoresis buffer for SDS-PAGE is SDS-tris-glycine, the so-called Laemmli buffer. An alternative is the tris-tricine-SDS system invented by Schaegger & Jagow. For native protein gels, tris-glycine buffer is the first choice.To initiate the polymerization, 100 µl of 10 % APS and 5-10 µl TEMED per 10 ml gel solution are added. Since the polymerization is very fast induced by TEMED and the radical initiator APS, the gel should be poured immediately. Cooling down the solution decelerates the polymerization process. APS (ammonium persulfate, A1142) is not very stable in aqueous solution (commonly a 10 % stock solution in water is prepared), but from experience, the solution can be stored at 2-8°C for several weeks, or at -20°C for months without losing its activity.
TEMED (tetramethylethylenediamine, A1148) enhances the polymerization of acrylamide and bisacrylamide by catalysing the formation of free radicals of APS. It is used in a concentration of 50 µl / 100 ml of gel solution. The stability of TEMED is very high (2 years), if contact to water is prevented.
Buffer solutions and other chemicals for polyacrylamide gel electrophoresis of proteins
|A4987||Tris buffer pH 6.8 (1M)||1M Tris buffer solution pH 6.8; 8X concentrated buffer solution for the stacking gel|
|A1411||Tris buffer pH 8.8 (1.5M)||1.5 M Tris buffer solution pH 8.8; 4X concentrated buffer solution for the resolving gel|
|A1415||SDS-Tris-Glycine buffer (10X)||Laemmli buffer, 10X concentrated. Contains 1.92 M glycine (A3707), 1 % SDS and 0.25 M tris (A1086). Available as 5X (A3376) solution as well.|
|A1418||Tris-Glycine buffer (10X)||Electrophoresis buffer for native protein gels; the 10X concentrated solution contains 1.92 M glycine (A3707) und 0.25 M tris (A1086). Also available as ready-to-use 1X (A3396) solution.|
|A3411||Tris-Tricine-SDS buffer (1X)||Alternative to the Laemmli system. Replacement of glycine by tricine improves the separation of 1 - 100 kDa proteins and peptides (discontinuous SDS-PAGE). Contains 17.9 g/L tricine (A1085), 0.1 % SDS and 12.1 g/L tris (A1086). Available as 5X (A3427) solution as well.|
|A3484||Loading buffer IX (for SDS gels)||2X loading buffer for SDS-PAGE; contains bromophenolblue, gycerol, SDS and 100 mM Tris⋅HCl (pH 6.8).|
|other buffer components
|A1142||Ammonium persulfate||APS; the recommended final concentration in acrylamide gels is 0.1 % (w/v). Prepare a 10 % (w/v) stock solution for an easy application.|
N,N,N',N'-Tetramethylethylenediamine; the recommended final concentration in acrylamide gels is 0.1 % (v/v).
|A0676||SDS - Solution 10 %||Sodium dodecylsulfate; also available as 20 % solution (A0675) or crystalline (e.g. A2572).|
ready-to-use Acrylamide solutions for SDS-PAGE
Save time and assure reproducible results: Our ready-to-use gel solutions for SDS-PAGE are your product of choice for an easy and fast gel preparation. AppliChem offers ready-to-use solutions for stacking and resolving gels according to Laemmli, based on an acrylamide/bisacrylamide ratio of 29 : 1 with 0.1% SDS and Tris.To prepare the acrylamide gel, 100 ml of each ready-to-use solution (see table below) is completed with 1 ml APS (from a 10% stock solution based on A1142) and 50 µl TEMED (A1148) to initiate the polymerization of the gel. For electrophoresis, a SDS-tris-glycine buffer (for example, A1415) is used.
Stacking and resolving gel ready-to-use solutions for SDS-PAGE (in Tris buffer)
|Acrylamide 4K - ready-to-use solutions according to Laemmli
|A0710||Acrylamide 4K - Ready-to-use solution for SDS-PAGE (7.5 %)||7.5% resolving gel solution; see A0711. Separation range: 35 - 95 kDa.|
|A0711||Acrylamide 4K - Ready-to-use solution for SDS-PAGE (10 %)||
10% resolving gel solution; acrylamide/bisacrylamide 4K quality, ratio 29 : 1, in 380 mM Tris (pH 8.8) and 0.1% SDS. Separation range: 15 - 70 kDa.
In addition to this resolving gel solution a stacking gel solution (A2529, A3759) is required.
|A0712||Acrylamide 4K - Ready-to-use solution for SDS-PAGE (12.5 %)||12.5% resolving gel solution; see A0711. Separation range: 14 - 57 kDa.|
|A0713||Acrylamide 4K - Ready-to-use solution for SDS-PAGE (15 %)||15% resolving gel solution; see A0711. Separation range: 12 - 45 kDa.|
|A2529||Acrylamide 4K - Stacking gel solution (4 %) for SDS-PAGE||4% stacking gel solution; acrylamide/bisacrylamide 4K quality, ratio 29 : 1, in 125 mM Tris (pH 6.8) and 0.1% SDS.|
|A3759||Acrylamide 4K - Stacking gel solution (5 %) for SDS-PAGE||5% stacking gel solution; see A2529.|
Protein markers and protein dyes
AppliChem offers a broad selection of prestained and unstained protein size standards for polyacrylamide gel electrophoresis. An advantage of prestained markers is that protein migration and transfer can be observed during electrophoresis and subsequent western blotting. Without any further staining procedure the marker proteins are visible on the membrane and simplify the evaluation of the best blotting conditions. For high-accuracy size estimation unstained markers should be used, since the covalent coupling of the dye may lead to changes in the running behavior.
Protein size standards for SDS-PAGE
|A5238||Protein Marker I (14 - 116)||7 bands; ready-to-use in loading buffer|
|A5418||Protein Marker II (6.5 - 200) prestained||8 bands; ready-to-use in loading buffer; the covalently prestained (blue) proteins are visible (on the gel and on membranes after transfer via western blot) without any further staining procedure|
|A4402||Protein Marker III (6.5 - 200)||8 bands; ready-to-use in loading buffer|
|A3993||Protein Marker IV (10 - 150)||8 bands; ready-to-use in loading buffer|
|A8359||Protein Marker V (10 - 175) prestained||11 bands; ready-to-use in loading buffer; bicolored (blue, pink) prestained protein bands.|
|A8889||Protein Marker VI (10 - 245) prestained||12 bands; ready-to-use in loading buffer; tricolored (blue, red, green) prestained protein bands.|
For non-specific staining of the protein bands on the polyacrylamide gel mainly Coomassie® Brilliant Blue R-250 is used. If higher sensitivity is required, silver nitrate staining is employed. Before or during the staining procedure, the proteins need to be fixed in the gel matrix. This is done by an aqueous mixture of ethanol and acetic acid or trichloroacetic acid that lead to protein denaturation and precipitation. For fixing of small basic proteins, formaldehyde is more suitable.
Protein dyes for staining of polyacrylamide gels
|A1092||Coomassie® Brilliant Blue R-250||One of the most commonly used stains for proteins in SDS-PAGE. The protein-dye complex has an absorption maximum at 549 nm. Per positively charged amino acid approximately 1.5 - 3 molecules of Coomassie® Brilliant Blue R-250 will be bound. The sensitivity is around 200-400 ng protein/band.|
|A3480||Coomassie® Brilliant Blue G-250||The main application of this dye is the Bradford assay (determination of protein concentration), but staining of proteins in polyacrylamide gels is possible as well.|
|A2176||Bismarck brown R||Increases the sensitivity of protein staining in polyacrylamide gels when used as a mixture with Coomassie® Brilliant Blue R-250. In this system, Bismarck brown is acting as an ion-pair reagent. The detection limit for BSA is about 25 ng.|
|A0822||Eosin Y||Rapid staining of proteins in low-pH, urea-containing polyacrylamide gels with eosin Y reveal similar staining intensities to those seen with Coomassie® R-250. High recoveries of total protein as well as enzymatic activity.|
|A1346||Eriochrome black T||
The mixed-dye staining method of proteins in polyacrylamide gels with Eriochrome black T in combination with Rhodamine B can detect as little as 10 ng of BSA within one hour and is more sensitive than Coomassie®-staining.
|A1401||Fast Green FCF||Alternative protein dye with an absorption maximum at 625 nm.|
|A2385||Congo red||Anionic dye, which binds to carboxymethylcellulose and proteins in acidic acetate buffer. The sensitivity is higher than for Ponceau S staining and slightly lower than Coomassie® Brilliant blue staining.|
|A3930||Rhodamine B||See Eriochrome black T.|
|A3972||Silver nitrate||Staining of proteins and nucleic acids with silver is one of the most sensitive non-specific detection methods (5-30 ng protein per band).
Caution: To avoid artefacts during the staining procedure wearing gloves is inevitable. Use deionised water and clean glassware (no plastics!).
|A1400||Stains all||Stains RNA (blue-violet, λmax 600 nm), DNA (blue, λmax 620 nm), proteins (red; for BSA: λmax 515 nm) and polysaccharides (λmax 600-640 nm) in agarose and polyacrylamide gels. The detection limit for proteins is approximately 100-200 ng protein per band.|
|A6810||Proteo-Dye Blue-Vis||Blue reversible protein dye with a detection limit of 3-5 ng/mm².|
|A7808||Proteo-Dye RuBPS||Fluorescence dye for protein detection in SDS- and 2-D-gels that provides the high sensitivity of silver staining without its drawbacks. Proteo-Dye RuBPS offers excellent contrast, good linearity and homogeneity. A minimal interference with MALDI-TOF analysis is observed and compatibility with MS/MS analysis is given. The photochemically stable dye is excited with UV-light of the wavelength 473/488 nm (blue laser). A 532 nm laser may be used as well, albeit with reduced efficiency.|
3. The history of electrophoresis - past and present
Charged molecules migrate in an electric field. This is the basic principle of electrophoresis, a method used in almost every biology lab today and fundamental for most separation techniques and analytic methods.
When we hear the term electrophoresis we will probably think of gel electrophoresis, agarose or acrylamide, especially SDS-PAGE – which is today the most popular and most widely used electrophoretic method in research worldwide. But the field of electrophoretic methods and applications is very broad and, in contrast to the now dominating techniques, the first steps of electrophoresis were performed in free solution without any kind of support medium. The early years
How did this story of success start? The basic theory of electrophoresis was developed more than 200 years ago, but it was only in the 1930s that Tiselius presented the so called moving boundary electrophoresis. This free solution electrophoresis technique was suitable to study the mobility of charged molecules. In the “Tiselius apparatus” the “moving boundaries” formed by electrophoretically migrating proteins were measured by the changes in light absorption or the refractive index. In contrast to the today’s methods, a complete separation of the components of a mixture was never achieved, no matter how long the experiment was conducted. Only partial analysis of the fastest and the slowest migration compounds was possible. In the 1950s, the moving boundary electrophoresis was outpaced by zone electrophoresis, a different principle of electrophoresis that was firstly described in 1939. In zone electrophoresis proteins, nucleosides, amino acids and other molecules were physically separated from each other with the help of a support medium. At the beginning, filter papers were used; but soon the method was strongly improved by alternative supports like cellulose acetate (Kohn 1957), starch (Smithies 1955), polyacrylamide (Raymond & Weintraub 1959) and agarose (Hjertén 1961). The support medium prevents the molecules from sedimentation and - in contrast to moving boundary electrophoresis - enables therefore complete separation.The invention of PAGE
With the 1960s, a novel era began in the field of electrophoresis. New techniques were introduced, already existing methods largely improved. Disc electrophoresis was developed as well as isoelectric focusing. Coomassie® Brilliant Blue was initially used as a dye to visualize protein bands after gel electrophoresis and SDS was identified to “mask” the net charge of proteins during polyacrylamide electrophoresis.
Five years after Raymond & Weintraub introduced polyacrylamide gels for protein electrophoresis this material was used by Ornstein and Davis as a support for a technique that is known and applied in almost every biochemical lab up to now: In 1964, they developed the disc (discontinuous) electrophoresis, which combines zone electrophoresis with isotachophoresis. By isotachophoresis - initially termed as ion migration method (Kendall & Crittenden 1923) or displacement electrophoresis (Martin 1942) - sharp boundaries between the sample constituents can be generated. Unlike zone electrophoresis, isotachophoresis is performed in a discontinuous buffer system, composed of a leading electrolyte of high mobility and a terminating or trailing ion of low mobility. After setting up the electric field, the molecules migrate at different speeds and therefore completely separate from each other forming stacks; the molecule with the highest mobility directly follows the leading ion, molecules with the lowest mobility migrate directly in front of the terminating electrolyte. Nothing special so far. However, the faster moving ions will lower the surrounding electrical field while the slower ones create a higher field. As a consequence, after separation, all molecules migrate with the same speed (and this is what the name isotachophoresis literally means: migration at equal speed): the initially faster migrating ions will be retarded by the surrounding weaker field; the slower moving ions will be accelerated by the stronger field around them. For this reason, the system has a self-sharpening effect; as soon as an ion diffuses out of its own band it will either be de-accelerated or accelerated and so forced backwards into its original band.
Back again to the principle of disc electrophoresis. By using 2 different areas of separation (stacking and resolving gel) and a discontinuous buffer system, Ornstein and Davis prevented the formation of protein aggregates during the entry into the gel matrix and supported the separation to well defined bands.
The stacking gel is characterized by a lower pH and larger pores than the adjacent resolving gel. Both gels contain only chloride ions (with Tris as a counter ion), while the electrode buffer contains only glycine. At first, the proteins are “stacked” and concentrated by the principle of isotachophoresis. Due to the large pores of the stacking gel, the size of the molecule does not influence their mobility. The net charge of glycine is almost zero at the pH of the stacking gel, therefore glycine functions as terminating ion. When the protein front reaches the border to the close-meshed resolving gel, the small glycine molecules pass through the proteins, enters the resolving area, becomes higher charged and moves together with the chloride ions in front of the protein fraction. As soon as the proteins are surrounded by the homogeneous buffer they start separating according to the principles of zone electrophoresis: now their mobility depends on their charge and size, which finally leads to a rearrangement of the protein-ranking.
When Laemmli in 1970 published his famous paper on T4 phage protein separation, he used Ornstein and Davis’ Tris-glycine-chloride buffer system. But instead of conducting a “native” PAGE, he added SDS, which was introduced by Shapiro et al. in 1967. In SDS-PAGE, the separation process is no longer based on the specific net charge of the proteins, but on their differences in molecular weight. The combination of SDS-PAGE with “Laemmli”-buffer is the most frequently used technique for separation of proteins till this date.
Another milestone in the development of gel electrophoresis was the isoelectric separation of proteins. The theory of isoelectric focusing was introduced by Svensson already in 1961, but only after Vesterberg successfully synthesized carrier ampholytes for generation of a continuous pH gradient, the technique of isoelectric focusing was born. In 1975, O’Farrell combined isoelectric focusing with SDS-PAGE; the so-called 2D-electrophoresis (or “protein-mapping”, since every protein is characterized by a specific position due to its intrinsic charge and mass) became an important tool for protein analysis.
A quantum jump in protein detection in acrylamide gels (especially 2D-electrophoresis) was the introduction of silver staining techniques by Merrill et al. 1979. Compared to the previously predominant Coomassie® staining, the sensitivity was strongly increased from micrograms to nanograms. In the same year, Towbin et al. performed the first Western Blot; he transferred SDS-PAGE-separated proteins to a nitrocellulose membrane.SDS-PAGE: Is there anything after Laemmli?
The Laemmli-coined performance of SDS-PAGE is present until now, so we have to ask if there are no alternatives. Is this the end of evolution in SDS-PAGE? No further techniques or improvements during the last 40 years? – Sure there are some!
There are a few classes of proteins that behave anomalously in SDS-PAGE: glycoproteins, strongly basic proteins (positively charged) and some hydrophobic transmembrane proteins. Regarding the highly hydrophilic glycoproteins, the usage of alkaline Tris-borate-EDTA buffer (Poduslo 1981) provides a solution. To separate histones, Panyim & Chalkley developed acid urea polyacrylamide gels (AU gels) in 1969. An alternative is the TAU gel, which additionally contains the non-ionic detergent Triton®. TAU gels are suitable for the identification of modifications of proteins such as acetylation and phosphorylation. Other highly charged proteins can be separated according to their size by using the cationic detergent cetyltrimethylammonium bromide instead of SDS (Eley et al. 1979).
For an improved separation of small peptides Schägger & Jagow used an alternative Tris-Tricine-buffer system for SDS-PAGE in 1987. Four years later the same researchers introduced a discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels. In their blue native electrophoresis, Coomassie instead of SDS is used to induce a charge shift on the proteins. In addition, aminocaproic acid and non-ionic detergents like Triton® X-100 serve to improve solubilization of the membrane proteins. The resulting dye-detergent-protein complexes are separated within a aminocaproic acid-containing PAG and, for studies on quaternary structure, resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE. For small membrane proteins, Coomassie® is proposed to be replaced by taurodeoxycholate.
The beauty of simplification - Ahn “Single gels”
In 2001, Ahn et al. introduced a simplified variant of the SDS-PAGE procedure that consists of only one “single gel” with an increased length for the separating gel. In Laemmli’s Tris-glycine gels, glycine serves as the slow moving ion. In contrast, Ahn single gels employ three amino acids for this purpose, namely glycine, serine, and asparagine. In Laemmli’s original protocol the separating gel works at pH value 8.8, whereas Ahn’s single gel works at pH 7.4. At such a mild alkaline pH value the hydrolysis of acrylamide is minimized. It improves stability and shelf life of acrylamide solutions and gels. Ahn gels do not contain any detergent but SDS is added to the running buffer to permit denaturing conditions during electrophoresis. In the original paper of Ahn et al., no comparison to other SDS-PAGE variants is shown. We wanted to study the differences in the performance of gels according to Ahn or Laemmli. To this end we conducted gel electrophoresis on 10 % polyacrylamide gels according to both of the protocols. Additionally, we investigated the transfer of proteins onto blotting membranes in a Western blot assays. Our results in brief:
The fist clear difference between Laemmli and Ahn gels is the length of the separation gel for the same type of gel cassette. Single gels according to Ahn make longer separation gels (Fig. 1, SDS-PAGE of total protein from HEK293T cell lysate using 10 % acrylamide gels according to Ahn et al. (A) and Laemmli (B). Coomassie® stained gels show about 20 prominent protein bands with different size distributions for both gel types. Migration patterns of proteins were different for both gel types. Proteins with molecular mass higher than 70 kD appear compressed on Laemmli Tris-glycine gels, whereas marker proteins were distributed linear over a wide range of sizes using an Ahn single gel.). However, the migration patterns for proteins differ between Laemmli Tris-gylcine and Ahn single gels at different molecular weight ranges. For proteins of 15 – 70 kilo Dalton (kD) 10 % Laemmli gels show the highest resolution whereas 10 % Ahn gels show a linear migration pattern for protein sizes ranging from 25 – 130 kD. When trying to separate a wide range of protein sizes on the same gel there are two options: Ahn single gels appear particularly well suited for medium and high molecular weight proteins. Laemmli Tris-glycine gels are best for small and medium sized proteins when used at the same concentration of acrylamide polymer.
We also studied the stability of acrylamide solutions according to Ahn et al. by storing them for a period of up to six months at 2-8°C. Polyacrylamide gels made from such solutions did not show any significant effect on quality even after prolonged storage. However, cast gels should be used within a few days since the pH value within the gel changes rapidly. This holds true for Laemmli Tris-glycine gels as well as for Ahn single gels.
3. Western Blotting.
For the transfer of proteins the performance varies depending on the molecular mass of the proteins (Fig. 2, Western blot of total protein from HEK293T cells after SDS-PAGE according to Ahn et al. (A) and Laemmli (B). Two different proteins were detected with specific antibodies: Tubulin and a 25 kD his-tagged protein. Expression of Tubulin was the same in all cells. The cellular (or proteasomal) protein degradation of the his-tagged protein was induced (+) or not induced (-) in the experiments. Tubulin was transferred more efficiently from Ahn gels to PVDF membranes than from Laemmli Tris-glycine gels, whereas the transfer of the 25 kD his-tagged protein was less efficient from equivalent Ahn single gels.). Tubulin, a protein of 66 kD, was transferred from Laemmli gels 55 ± 4 % less efficiently compared to Ahn single gels. In contrast, the transfer of a 25 kD his-tag protein was less efficient from Ahn gels by 32 ± 12 % (n=4). Overall, the transfer of small proteins is better using Laemmli’s Tris-glycine gels, while Ahn single gels performed better for proteins of higher molecular mass.
Interviewed users liked the fast and convenient procedure according to Ahn et al. The protocol is easily adopted by laboratories already using the “classical” Laemmli protocol since all apparatus, buffers and solutions are the same for both SDS-PAGE procedures. Other, more skeptical authors also evaluated Ahn’s single gel system and finally confirmed the good performance. According to G. Fritz (from the Univ. of Zürich, in Rehm 2007) additional benefits of Ahn gels are: 1. they run quite beautifully (do not tend to produce “smilies”), 2. gels work at higher polyacrylamide concentrations (up to 18 %), 3. they avoid the slimy stacking gel.Perspectives
In research, new methods are constantly invented creating new insights. Some of the methods such as the gel electrophoresis withstand the test of time. Classical methods were improved, adjusted, and new features were added over the years. AppliChem is always striving to explore new or improved application protocols to share promising products with the customers.