Although essentially an analytical technique, PAGE does of course achieve fractionation of a protein mixture during the electrophoresis process. It is possible to make use of this fractionation to examine further individual separated proteins. The first step is to transfer or blot the pattern of separated proteins from the gel onto a sheet of nitro cellulose paper or polyvinylidene fluoride (PVDF) membrane, hereafter referred to as membrane. The method is known as protein blotting, or Western blotting by analogy with Southern blotting, the equivalent method used to recover DNA samples from an agarose gel. Transfer of the proteins from the gel to nitrocellulose is achieved by a technique known as electroblotting. In this method, a sandwich of gel and nitrocellulose is compressed in a cassette and immersed, in buffer, between two parallel electrodes ( Figure 1).

Fig1. Diagrammatic representation of electroblotting. The gel to be blotted is placed on top of a sponge pad saturated in buffer. The nitrocellulose sheet is then placed on top of the gel, followed by a second sponge pad. This sandwich is supported between two rigid porous plastic sheets and held together with two elastic bands. The sandwich is then placed between parallel electrodes in a buffer reservoir and an electric current passed. The sandwich must be placed such that the immobilising medium is between the gel and the anode for SDS polyacrylamide gels, because all the proteins carry a negative charge.

A current is passed at right angles to the gel, which causes the separated proteins to migrate out of the gel and into the nitrocellulose sheet. The nitrocellulose with its transferred protein is referred to as a blot. Once transferred onto nitrocellulose, the separated proteins can be examined further. This involves probing the blot, usually using an antibody to detect a specific protein. The blot is first incubated in a protein solution, for example 10% (w/v) bovine serum albumin, or 5% (w/v) non-fat dried milk (known as the so-called blotto technique), which will block all remaining hydrophobic binding sites on the nitrocellulose sheet. The blot is then incubated in a dilution of an antiserum (primary antibody) directed against the protein of interest. This immunoglobulin G (IgG) molecule will bind to the blot if it detects its anti gen, thus identifying the protein of interest. In order to visualise this interaction, the blot is incubated further in a solution of a secondary antibody, which is directed against the IgG of the species that provided the primary antibody. For example, if the primary antibody was raised in a rabbit, then the secondary antibody would be anti-rabbit IgG. This secondary antibody is appropriately labelled so that the inter action of the secondary antibody with the primary antibody can be visualised on the blot. Anti-species IgG molecules are readily available commercially, with a choice of different labels attached. One of the most common detection methods is to use an enzyme-linked secondary antibody (Figure 2); the principle behind the use of enzyme-linked antibodies to detect antigens in blots is highly analogous to that used in enzyme-linked immunosorbent assays ( ELISAs). In this case, following treatment with enzyme-labelled secondary antibody, the blot is incubated in enzyme–substrate solution, when the enzyme converts the substrate into an insoluble coloured product that is precipitated onto the nitrocellulose. The presence of a coloured band therefore indicates the position of the protein of interest. By careful comparisons of the blot with a stained gel of the same sample, the protein of interest can be identified. The enzyme used in enzyme-linked antibodies is usually either alkaline phosphatase, which converts colourless 5-bromo-4-chloro-indolylphosphate (BCIP) substrate into a blue product, or horseradish peroxidase, which, with H₂O₂ as a substrate, oxidises either 3-amino-9-ethylcarbazole into an insoluble brown product, or 4-chloro-1-naphthol into an insoluble blue product. An alternative approach to the detection of horseradish peroxidase is to use the method of enhanced chemiluminescence (ECL). In the presence of hydrogen peroxide and the chemiluminescent substrate luminol (Figure 3), horseradish peroxidase oxidises the luminol with concomitant production of light, the intensity of which is increased 1000-fold by the presence of a chemical enhancer. The light emission can be detected by exposing the blot to a photographic film. Corresponding ECL substrates are available for use with alkaline-phosphatase-labelled antibodies.

Fig2. The use of enzyme-linked second antibodies in immunodetection of protein blots. First, the primary antibody (e.g. raised in a rabbit) detects the protein of interest on the blot. Second, enzyme-linked anti-rabbit IgG detects the primary antibody. Third, addition of enzyme substrate results in coloured product deposited at the site of the protein of interest on the blot.

Fig3. The use of enhanced chemiluminescence to detect horseradish peroxidase.

Although enzymes are commonly used as markers for second antibodies, other markers can also be used. These include:

• ¹²⁵I-labelled secondary antibody: Binding to the blot is detected by autoradiography.

• Fluorescein-labelled secondary antibody : The fluorescent label is detected by exposing the blot to ultraviolet light.

• ¹²⁵I-labelled protein A: Protein A is purified from Staphylococcus aureus and specifically binds to the Fc region of IgG molecules. ¹²⁵I-labelled protein A is therefore used instead of a second antibody, and binding to the blot is detected by autoradiography.

• Biotinylated secondary antibodies: Biotin is a small-molecular-weight vitamin that binds strongly to the egg protein avidin (K_d = 10⁻¹⁵). The blot is incubated with biotinylated secondary antibody, then incubated further with enzyme-conjugated avidin. Since multiple biotin molecules can be linked to a single antibody molecule, many enzyme-linked avidin molecules can bind to a single biotinylated antibody molecule, thus providing an enhancement of the signal. The enzyme used is usually alkaline phosphatase or horseradish peroxidase.

• Quantum dots: These are engineered semiconductor nanoparticles, with diameters of the order of 2–10 nm, which fluoresce when exposed to UV light. Quantum dot nano crystals comprise a semiconductor core of CdSe surrounded by a shell of ZnS. This crystal is then coated with an organic molecular layer that provides water solubility, and conjugation sites for biomolecules. Typically, therefore, secondary antibodies will be bound to a quantum dot, and the position of binding of the secondary antibody on the blot identified by exposure to UV light.

In addition to the use of labelled antibodies or proteins, other probes are sometimes used. For example, radioactively labelled DNA can be used to detect DNA-binding proteins on a blot. The blot is first incubated in a solution of radiolabelled DNA, then washed, and an autoradiograph of the blot made. The presence of radioactive bands, detected on the autoradiograph, identifies the positions of the DNA-binding proteins on the blot.

اخر الاخبار

اخبار العتبة العباسية المقدسة

مؤسّسة الوافي تُهدي مكتبةَ مسجد الكوفة نسخةً من موسوعة فتوى الدفاع الكفائي

المجمع العلمي يقيم برنامج (سراج المنتظرين) في محافظة المثنى

قسم الشؤون الفكرية ينظم ندوة فكرية عن أبعاد نهضة الإمام الصادق (عليه السلام)

العتبة العباسية المقدسة تناقش تكثيف جهودها الإغاثية لدعم الشعبين الإيراني واللبناني

المزيد

الآخبار الصحية

دواء جديد يحمي القلب ويعزز علاج السرطان في آن واحد

سر غذائي بسيط لحماية القلب مع التقدم في العمر

علامات تشير إلى اختلال عمل الغدة الدرقية

السمنة واللقاحات.. دراسة تكشف تأثيراً سلبياً

المزيد

الآخبار التكنلوجية

البعوض في آيسلندا إشارة مبكرة على تحولات بيئية في القطب الشمالي

صاروخ يطلق قمرا اصطناعيا في مدار خاطئ

"أوبن إيه آي" تطلق نموذجا للعلوم الحيوية واكتشاف الأدوية

تفوق على البشر.. روبوت يحطم الرقم القياسي لسباق نصف ماراثون !

المزيد

مواضيع ذات صلة

Detection, Estimation and Recovery of Proteins in Gels

Cellulose Acetate Electrophoresis

Other Methods of Protein Purification

Chromatographic Methods for Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production

Introduction to Immunological methods