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Western Blot: a Tool in the Biomedical Field

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Western Blot: a Tool in the Biomedical Field www.medigraphic.org.mx General interest Vol. 6, No. 3 September-December 2017 Western blot: a tool in the pp 128-137 biomedical field Western blot: una herramienta en el área biomédica Karina Martínez-Flores,* Ángel Tonatiuh Salazar-Anzures,* Javier Fernández-Torres,* Carlos Pineda,* Carlos Alberto Aguilar-González,* Alberto López-Reyes* * Laboratorio de Líquido Si- novial, Instituto Nacional de Rehabilitación. Mailing address: Karina Martínez-Flores Abstract Calzada México-Xochimilco Núm. 289, Col. Arenal de Guadalupe, 14389, The Western blot technique, also called immunoblotting, is a high-sensitivity and semi-quantitative Ciudad de México, México. molecular technique that allows the analysis of a specific protein or protein profile. This is a Phone: (55) 5999 1000, 19502 technique for separating proteins according to their charge and molecular weight in polyacrylamide E-mail: [email protected] gels and their subsequent transference to a solid membrane, in which a specific antibody detects Received: September 08, 2016. the protein. Four decades after its creation, the Western blot technique has gained an essential role Accepted: March 03, 2017. in the diagnosis of various medical conditions, as well as an everyday tool in biomedical research. This article can be read in its full Resumen version in the following page: http://www.medigraphic.com/rid La inmunotransferencia de proteínas, o Western blot, es una técnica molecular semicuantitativa de alta sensibilidad que permite la inmunodetección específica de una proteína o perfil de Key words: proteínas. Esta técnica se basa en la separación de proteínas de acuerdo con su carga y peso Western blot, molecular en geles de poliacrilamida y su posterior transferencia a una membrana sólida, en immunodetection, la que un anticuerpo específico detecta la proteína de interés. Cuatro décadas después de electrophoresis, protein, su creación, la técnica de Western blot ha ganado un papel importante en el diagnóstico de immunoblot, antibody. varias condiciones médicas, además de constituir una herramienta de cada día en la inves- tigación biomédica. Palabras clave: Western blot, inmunodetección, electroforesis, proteína, inmunoblot, anticuerpo. Introduction attached to a developer that identifi es the protein of interest (Figure 1A).1-3 The WB emerged in 1970, when The Western blot (WB) analysiswww.medigraphic.org.mx is an analytical Ulrich Laemmli used the principle of molecule separation technique used to detect proteins. The technique is through an electrical gradient using polyacrylamide gels. based on the separation of proteins by molecular weight At the end of this decade, Towbin (1979) described (MW) and electric charge using a gel electrophoresis. the transfer of proteins from polyacrylamide-urea gels Afterwards, the proteins are transferred to a synthetic to nitrocellulose sheets, being Burnette two years membrane and they are exposed to antibodies of later, who employed the term Western blot (WB) for interest (primary antibodies). Most of the time, the the technique using polyacrylamide gels and sodium primary antibody must be coupled to a second antibody dodecyl sulfate (SDS-PAGE).4-7 Nowadays, the WB 128 Investigación en Discapacidad Karina Martínez-Flores et al. Load the proteins The proteins are separate The proteins are transferred from Then membrane is Then the membrane is incubated through electrophoresis and the gel to a synthetic membrane, incubated with with the secondary antibody coupled it’s verify with Coomassie blue migration is confirmed by red the primary antibody to a peroxidase or phosphatase enzyme A ponceau that will reveal the protein of interest Figure 1. Load samples Running (A) The steps of the western Molecular weight blot. a) Loading the protein marker into the gel, separation by electrophoresis, transfer and immunomarking. (B) The protein sample is loaded using a micropipette for Tracking dye precision (20-40 μg). (C) The separated proteins are visualized with a tracking B C dye (bromophenol blue) and molecular weight marker. has become a key technique in the biomedical fi eld and slowly, avoiding protein denaturation and protein for diagnosing infectious, autoimmune, rheumatic and complex breakdown; this method is recommended for oncologic diseases.3,4,8 This review is centralized in assays involving protein structure or protein activity. describe and analyze the main characteristics of the WB The zwitterionic detergents,-like 3 [(3-cholamidopropyl) and the importance of this technic in the biomedical fi eld. dimethylammonium] -1-propanesufonate can be used without affecting the net charge or the charge Cell lysis to extract protein of the solubilized protein. One of the most widely used buffers for its effi ciency solubilizing proteins The WB is a versatile technique that detects proteins is radioimmunoprecipitation assay (RIPA), which from animal and herbal tissues, cell cultures, bacteria contains 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and yeast. In order to obtain trustworthy results, a good 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% protein extraction is required, which can be performed SDS, 5 mM EDTA, and a commercial inhibitor of by mechanical and chemical methods. Within the proteases.10 In HT-29, the colon cancer cell line, this mechanical methods, the most used are sonication, reagent has showed a high effi ciency solubilizing homogenization using abrasives, and disintegration cytoplasmic proteins.11 Besides RIPA, there are buffers with glass or metallic beads.9 The chemical methods for extracting cytoplasmic proteins, such as Tris-Triton include the use of buffers capable of solubilizing the (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM proteins, since they contain ionic detergents such EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, as sodium dodecyl sulfate (SDS),www.medigraphic.org.mx deoxycholate and 0.5% deoxycholate) and the 20 mM Tris-HCl pH 7.5 for cetyl trimethylammonium bromide (CTAB). It is also cytoplasmic proteins. The purpose of using lysis-buffer possible to use other detergents, such as non-ionic solutions supplemented with proteases inhibitors is to or zwitterionic chemicals, and the choice depends on maintain the protein structures. Finally, after the lysis the speed of the lytic effect, as well as the extraction process, the sub-cellular fractions are separated trough effi ciency desired. For example, SDS can lyse cells in differential centrifugations, at the end; it is possible to seconds, but it can denature the proteins. Triton X-100 obtain membrane proteins in the pellet and soluble is a non-ionic detergent that can lyse cells smoothly proteins in the supernatant.4,12 Volume 6 Number 3 September-December 2017 129 Western blot: a tool in the biomedical field Protein quantification than 50 kDa), whereas gels with a lower percentage of acrylamide (< 10% T) are recommended for Before performing the electrophoresis, the protein separating higher molecular weight proteins (greater content needs to be quantifi ed in order to homogenize than 100 kDa).16-18 Adding ammonium persulfate and the amount deposited in the well of the gel and to tetramethylethylenediamine (TEMED) generates free determine the protein concentration. A large amount radicals that accelerate the acrylamide polymerization. of protein can saturate the immunodetection and yield The versatility of the running buffers for polyacrylamide unspecifi c results.13 gels provides a fast method to separate proteins in There are different methods to quantify proteins. order to identify them.18 The most used are Lowry, Bradford and bicinchoninic acid (BCA). These colorimetric assays are based on Types of gels the color change when some protein amino acids react to different reagents.13,14 Protein separation through polyacrylamide gels can be performed under naive or denaturing conditions. LOWRY PROTEIN ASSAY The non-denaturing polyacrylamide gels (ND-PAGE) keep the protein structure in a tridimensional shape This method is characterized by the use of Follin and the separation is based on the electric charge, reagent and copper, it is detected in a wavelength of size and shape. In order to maintain the protein 750 nm, and the color intensity is based on the protein structure, a non-reductive, non-denaturant buffer concentration.15 solution is used, such as tri-glycine within a pH range between 8.3 and 9.5, or tris-borate at a pH of 19 BRADFORD PROTEIN ASSAY 7.0 to 8.5, and tris-acetate with a pH of 7.2 to 8.5. In contrast, in the denaturing polyacrylamide gels This method uses Coomasie Brilliant Blue dye, which (SDS-PAGE), the protein structures are dissociated in reacts in the presence of proteins, and the color change peptides using a combination of an anionic detergent is detected in the wavelength range of 465-595 nm.15 (SDS), a reducing agent (beta-mercaptoethanol), and heat. SDS binds and provides negative charge BICINCHONINIC ACID ASSAY to proteins and breaks the hydrophobic interactions, whereas beta-mercaptoethanol and the heat break The protein assay with bicinchoninic acid (BCA) is the disulfi de bridges (Cys-S-S-Cys) and thiol groups highly sensitive and combines the reaction of proteins (Cys-SH) present in the polypeptide chains, ensuring with Cu2+ ions in an alkaline method in the presence of protein dissociation (Figure 2A).4,16,18,20 Once the gel is a reagent that detects Cu+ ions with a high sensitivity, polymerized, a suitable concentration of protein (20- known as BCA. Its variation is minimum. It has been 40 μg) is deposited in the well created
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