SDS–polyacrylamide gel electrophoresis ( SDS–PAGE) is the most widely used method for analysing protein mixtures qualitatively. It is particularly useful for monitoring protein purification and, because the method is based on the separation of proteins according to size, it can also be used to determine the relative molecular mass of proteins. SDS (H₃C-(CH₂)₁₀ -CH₂ -OSO₃ ^–Na +) is an anionic detergent. For denaturing- reducing SDS-PAGE , samples to be run on SDS–PAGE are firstly boiled for 5 min in sample buffer containing β-mercaptoethanol or dithiothreitol ( DTT) and SDS. Mercaptoethanol or DTT reduce any disulfide bridges present that are holding together the protein tertiary structure, and the SDS binds strongly to the thermally denatured protein (albeit SDS on its own also causes protein denaturation to some extent). Each protein in the mixture is therefore fully denatured by this treatment and opens up into a rod-shaped structure with a series of negatively charged SDS molecules along the polypeptide chain. On average, one SDS molecule binds for every two amino-acid residues. The original native charge on the molecule is therefore completely swamped by the negatively charged SDS molecules. The rod-like structure remains, as any rota tion that tends to fold up the protein chain would result in repulsion between nega tive charges on different parts of the protein chain, returning the conformation back to the rod shape. The sample buffer also contains an ionisable tracking dye, usually bromophenol blue, that allows the electrophoretic run to be monitored, and sucrose or glycerol, which gives the sample solution density thus allowing the sample to settle easily through the electrophoresis buffer to the bottom when injected into the loading well (see Figure 1 ). Once the samples are all loaded, a current is passed through the gel. In conventional discontinuous gel electrophoresis , the samples to be separated are not in fact loaded directly into the main separating gel. When the main separating gel (normally about 5 cm long) has been poured between the glass plates and allowed to set, a shorter (approximately 0.8 cm) stacking gel is poured on top of the separating gel and it is into this gel that the wells are formed and the proteins loaded. The purpose of this stacking gel is to concentrate the protein sample into a sharp band before it enters the main separating gel. This is achieved by utilising differences in ionic strength and pH between the electrophoresis buffer (pH 8.8) and the stacking gel buffer (pH 6.8) and involves a phenomenon known as isotachophoresis . The stacking gel has a very large pore size (4% acrylamide), which allows the proteins to move freely and concentrate, or stack, under the effect of the electric fi eld. The band-sharpening effect relies on the fact that negatively charged glycinate ions (in the electrophoresis buffer) have a lower electrophoretic mobility than do the protein–SDS complexes, which, in turn, have lower mobility than the chloride ions (Cl⁻ ) of the loading buffer and the stacking gel buffer. When the current is switched on, all the ionic species have to migrate at the same speed, otherwise there would be a break in the electrical circuit. The glycinate ions can move at the same speed as Cl⁻ ions only if they are in a region of higher fi eld strength. Field strength is inversely proportional to conductivity, which is proportional to concentration. The result is that the three species of interest adjust their concentrations so that [Cl⁻] > [protein–SDS] > [glycinate]. There is only a small quantity of protein–SDS complexes, so they concentrate in a very tight band between glycinate and Cl⁻ boundaries. Once the glycinate reaches the separating gel it becomes more ionised in the higher pH environment and its mobility increases. Thus, the interface between glycinate and Cl⁻ leaves behind the protein–SDS complexes, which are left to electrophorese at their own rates. The negatively charged protein–SDS complexes now continue to move towards the anode, and, because they have the same charge per unit length, they travel into the separating gel under the applied electric fi eld with the same mobility. However, as they pass through the separating gel the proteins separate, owing to the molecular sieving properties of the gel. Quite simply, the smaller the protein the more easily it can pass through the pores of the gel, whereas large proteins are successively retarded by frictional resistance due to the sieving effect of the gels. Being a small molecule, the dye bromophenol blue migrates in a non-retarded fashion and therefore indicates the electrophoresis front. When the dye reaches the bottom of the gel, the current is turned off, and the gel is removed from between the glass plates and shaken in an appropriate stain solution (usually Coomassie Brilliant Blue) and then washed in destain solution. The destain solution removes unbound background dye from the gel, leaving stained proteins visible as blue bands on a clear background. A typical minigel would take about 1 h to prepare and set, 40 min to run at 200 V and have a 1 h staining time with Coomassie Brilliant Blue. Upon destaining, strong protein bands would be seen in the gel within 10–20 min, but overnight destaining is needed to completely remove all background stain. Vertical slab gels are invariably run, since this allows up to 10 different samples to be loaded onto a single gel. A typical SDS–polyacrylamide gel is shown in Figure2.

Fig1. Photograph showing an assembled SDS-PAGE minigel. Nine wells that have been loaded can be identified by the blue dye (bromophenol blue) that is incorporated into the loading buffer. The outermost left lane was loaded with a protein marker.

Fig2. A typical Coomassie-stained SDS polyacrylamide single gel. The first track shows a protein marker, whereas tracks 2–10 were loaded with elution fractions and flow-through (unbound sample) from a protein chromatography experiment.

Typically, the separating gel contains between 5 and 15% polyacrylamide. This gives a gel of a certain pore size in which proteins of relative molecular mass (M_r) 10 000 move through the gel relatively unhindered, whereas proteins of M_r = 100 000 can only just enter the pores of this gel. Gels of 15% polyacrylamide are therefore useful for separating proteins in the range M_r = 100 000 to 10 000. However, a protein of M_r = 150 000, for example, would be unable to enter a 15% gel. In this case a larger-pored gel (e.g. a 10% or even 7.5% gel) would be used so that the protein could now enter the gel and be stained and identified. It is obvious, therefore, that the choice of gel to be used depends on the size of the protein being studied. The fractionation range of different percentage acrylamide gels is shown in Table 1, illustrating, for example, that in a 10% polyacrylamide gel, proteins greater than 200 kDa in mass cannot enter the gel, whereas proteins with relative molecular mass in the range 200 000 to 15 000 will separate. Proteins of M_r = 15 000 or less are too small to experience the sieving effect of the gel matrix, and all run together as a single band at the electrophoresis front.

Table1. The relationship between acrylamide gel concentration and protein fractionation range

More recently, a system comprising only a single gel, as opposed to the conventional stacking and separation gels, has been introduced. These single gels are simpler and more convenient to prepare, as the need for two different gel layers has been eliminated. The performance of these gels in PAGE, staining/destaining, and electroblotting is virtually indistinguishable from that of discontinuous gel systems. Different from conventional gels, the matrix of single gels contains three amino acids (serine, glycine and asparagine), yet no SDS. This provides a further advantage of the single gel system, because a prepared gel can be used either as SDS, SDS/denaturing or native gel, simply depending on the choice of sample preparation and running buffer.

The M_r of a protein can be determined by comparing its mobility with those of a number of standard proteins of known M_r that are run on the same gel. By plotting a graph of distance moved against log M_r for each of the standard proteins, a calibration curve can be constructed. The distance moved by the protein of unknown M_r is then measured, and then log M_r and hence M_r can be determined from the calibration curve.

SDS–gel electrophoresis is often used after each step of a purification protocol to assess the purity or otherwise of the sample. A pure protein should give a single band on an SDS–polyacrylamide gel, unless the molecule is made up of two unequal subunits. In the latter case, two bands, corresponding to the two subunits, will be seen. Since only submicrogram amounts of protein are needed for the gel, very little material is used in this form of purity assessment and at the same time a value for the relative molecular mass of the protein can be determined on the same gel run, with no more material being used.

اخر الاخبار

اخبار العتبة العباسية المقدسة

بالتعاون مع قسم الشعائر.. المواكب العزائية تحيي ذكرى شهادة الإمام جعفر الصادق (عليه السلام) في كربلاء

المجمَع العلمي يقيم الاختبار المركزي لطلبة الحفظ في بغداد

قسم الشؤون الطبية ينظّم زيارة لدعم الأطفال المصابين بالسرطان في مستشفى الإمام الحسن (عليه السلام)

قسم الشؤون الفكرية يطلق مسابقة (حامل اللواء) بنسختها الثانية في بغداد

المزيد

الآخبار الصحية

بديل طبيعي للفلورايد يحمي أسنانك من التسوس

تحذير للرجال.. أطعمة شائعة الاستهلاك قد تضر بصحة البروستات

اكتشاف بديل طبيعي لـ"أوزمبيك" يخفض الوزن دون آثار مزعجة

خبراء يكشفون لماذا تنام النساء أكثر من الرجال ؟

المزيد

الآخبار التكنلوجية

الأرض "تومض" ليلا!.. كوكبنا ازداد سطوعا بنسبة 16% في غضون 8 سنوات

أحد أخطر البراكين العملاقة على وجه الأرض يمتلئ بالصهارة!

علماء يطورون تقنية جديدة تُسرّع التئام الجروح عبر تنشيط خلايا الجلد

بعد مهمة "أرتميس 2".. أوروبا تخطط لإرسال رواد فضاء إلى القمر

المزيد

مواضيع ذات صلة

Androgen Biosynthesis

Androgens and Related Molecules

Two-Dimensional Polyacrylamide Gel Electrophoresis

Isoelectric Focussing Gels

Native (Buffer) Gels

Transition Metals are Essential for Health

Individual Regulatory Events Combine to Form Sophisticated Control Networks

Regulation of Enzyme Quantity

Biochemical Evaluation of Cobalamin and Folate Deficiencies

Compartmentation Ensures Metabolic Efficiency & Simplifies Regulation

Regulation of Metabolite Flow can be Active or Passive

Catabolism of Catecholamines