¡Descarga Practicas metodologia y más Ejercicios en PDF de Metodología de Investigación solo en Docsity!
PRACTICE 6: DENATURING POLY-ACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) ANALYSIS OF PROTEIN SAMPLES
Aim: to separate and visualize proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and estimate their molecular weight. To monitor the development of a purification process.
BACKGROUND
Electrophoresis is one of the most widespread techniques for the separation and analysis of biological macromolecules as a function of their size and charge. Protein samples are usually analyzed using polyacrylamide gels, which provide suitable pore sizes for the separation of most proteins. PAGE electrophoresis can be performed either in native or denaturing conditions.
Unlike nucleic acids, proteins do not display a general net charge, but they rather possess different patches of charged and uncharged residues along the molecule. Besides, there are strong differences in 3D conformation among proteins that ultimately result in different electrophoretic behavior for each of them relying more on their compactness than on their true size.
Denaturing electrophoresis intends to normalize the electrophoretic mobility as a function of protein size. This is achieved by the action of SDS (sodium dodecylsulphate), an amphipathic surfactant which binds proteins through its hydrocarbon tail and coats the protein chain with surfactant molecules imparting an even distribution of charge per unit mass. Binding of SDS thus induces the unfolding of the polypeptide chain and provides a consistent mass/charge ratio among proteins, allowing the separation only as a function of the mass of the protein.
Sample preparation for SDS-PAGE is usually very simple since all the required components are contained in the so-called “Laemmli sample buffer”. In addition to SDS, it usually contains a reducing agent like β-mercaptoethanol that reduces disulfide bonds and helps achieving a complete denaturation. Other basic components are bromophenol blue, used as a tracking dye, and glycerol to increase the density and facilitate sample loading. Once mixed with the sample buffer, samples will be boiled at 95-100ºC to favor denaturation. Denatured, negatively-charged proteins will now move towards the positive pole during electrophoresis and their different mobility, based only on their molecular weight, will be exaggerated due to their movement across a sieving matrix.
The most suitable matrix for the separation of protein mixtures is polyacrylamide, a quite inert synthetic polymer that can be adjusted to a variety of pore sizes by altering the concentration of acrylamide monomers and that of the crosslinker bisacrylamide. Gels for SDS-PAGE are formed by mixing a solution of acrylamide-bisacrylamide with a buffer (usually Tris at a suitable pH) and SDS followed by the addition of ammonium persulfate (APS), a source of free radicals that initiates polymerization, and TEMED (N,N,N’,N’-tetramethylethane-1,2-diamine), used as a catalyst. Gels are polymerized between two glass plates with a very thin separation (0.75-1.5 mm), and with a comb inserted at the top to create the wells into which the samples will be loaded. In order to achieve a better separation, the wells are usually created on a smaller gel termed “stacking” gel, polymerized on top of the larger “resolving” gel. This is called a discontinuous system and basically allows narrowing the starting point to achieve thinner and better-resolved protein bands. Polyacrylamide gels are run in vertical electrophoresis systems and are stopped once the tracking dye reaches the bottom of the resolving gel.
EXPERIMENTAL PROCEDURE
A. Preparation of gels
I this session we will a discontinuous gel system, composed of a resolving gel and a stacking gel. Volumes of the reagents required to prepare each of these gels are shown in the following table:
Resolving gel (12%) Stacking gel (5%) 40% Acrylamide-Bisacrylamide 1.5 ml 0.260 ml
Water 2.15 ml 2.315 ml
1 M Tris-HCl pH 8.8 1.25 ml - 20 % SDS 0.025 ml 0.015 ml
10% Ammonium persulfate 0.050 ml 0.015 ml
TEMED 0.005 ml 0.005 ml 1 M Tris-HCl pH 6.8 - 0.375 ml
carefully pour the solution between both glass plates up to 1-2 cm below the level that will be later occupied by the well-forming comb. Place some water-saturated isobutanol on top of the gel and let the system polymerize at room temperature. This will take some time.
Once it has polymerized, remove the isobutanol, wash with distilled water and prepare the stacking gel solution. Pour this solution over the resolving gel until the space between the glass plates has been filled and place a 10-well comb avoiding the formation of bubbles between the comb and the gel. Allow polymerization at room temperature.
B. Preparation of the sample
Prepare each protein fraction in one Eppendorf tube, following the instructor´s guidelines 1) Lysozyme purification analysis : samples will contain 5 μl of the protein sample (F0, F1, W1, W2, E1) + 7 μl mQ water + 4 μl 4X Laemmli sample buffer. 2) Commercial protein samples: mix 4 μl of each protein (Lysozyme 4 mg/ml, Catalase 4 mg/ml, BSA 2mg/ml) + 8 μl mQ water + 3 μl 4X Laemmli sample buffer. Heat samples at 95ºC for 3 min, then put them on ice for 1 min
C. Gel load and electrophoresis running.
Upon polymerization of the stacking gel, remove the comb taking care not to damage the wells. Assemble the gel in the electrophoresis chamber following the instructor´s guidelines. Add electrophoresis buffer (TGS) The instructor will load the molecular weight markers in each gel. Then, load the wells carefully with the samples in the following order: Molecular weight marker - F0-F1-W1-W2-E1 – Lysozyme – Catalase - BSA Close the chamber and run the electrophoresis at a 150 V constant voltage until bromophenol blue is approximately 1 mm away from the lower end of the gel. Please, take care that the limit of the blue does not escape from the acrylamide gel to the buffer
D. Staining the gels with Coomassie blue.
Disassemble the gel and stain with methanol/acetic acid/H2O (5/1/5) with Coomassie blue (1 g/L). Staining step will be incubated at room temperature with mild shaking overnight Next day, destain the gel in methanol/acetic acid/ H 2 O (10/10/80).
Analyse your results
