Exercise No. 9: Protein Electrophoresis (SDS-PAGE) PDF

WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 9: Protein electrophoresis via the SDS-PAGE method Theoretical Part Student: ✓ understands the principles of protein electrophoretic separation, ✓ knows the structure and proper...

WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 9: Protein electrophoresis via the SDS-PAGE method Theoretical Part Student: ✓ understands the principles of protein electrophoretic separation, ✓ knows the structure and properties of proteins. Electrophoresis is a method of separating molecules in which, under the influence of an applied electric field, macromolecules move with an uneven electric charge. For the separation of proteins, polyacrylamide gel electrophoresis is mainly used. In this electrophoresis, the electrophoretic carrier is polyacrylamide, which allows for: separation of protein molecules in the range of 5 kDa - 300 kDa, separation of polynucleotides sized 5 - 2,000 base pairs, staining of separated DNA fractions through silver staining, providing distinct, visible, and enduring images. it is cost-effective. Poliacrylamide Gels These gels are prepared from a solution of acrylamide monomers and cross-linking agents. The most common cross-linking agent is N,N-methylenebisacrylamide (bis- acrylamide). The polymerization reaction, essentially a free-radical polymerization reaction, can be initiated chemically or photochemically. The cross-linking density and pore sizes can be regulated by choosing the concentrations of acrylamide and bis-acrylamide. Attention! Acrylamide in its monomeric form is a potent neurotoxin. Amino acids, peptides, and proteins can act as both bases and acids, exhibiting amphoteric (amphiprotic or amphoteric) properties, meaning they possess a net positive or negative electric charge depending on environmental conditions. The amphoteric characteristic results from the properties of amino acid residues (they can simultaneously be both basic and acidic). Most of the protein's electric charge comes from the pH-dependent ionization of the side chains of carboxyl groups and amino groups (-COOH ↔ COO- + H+; -NH2 + H+ ↔ -NH3+). That is why, the resultant electric charge of a protein molecule depends on the pH value of the solution and it is possible to find such a pH value at Strona 1 z 6  WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 which this resultant charge is equal to zero (isoelectric point = pI). Each protein has its own characteristic isoelectric point. The strength of the electric field's interaction during protein electrophoresis on the electric charge of the ion is proportional to the intensity of that field E [V/m], and the intensity of that field, in turn, is proportional to the voltage U [V] applied to the electrodes. Protein electrophoresis can be performed using polyacrylamide gels. These gels are prepared from a solution of acrylamide monomers and cross-linking agents. Acrylamide in its monomeric form is a potent neurotoxin, and even after the polymerization process, it poses a serious health hazard due to the remnants of free monomers within the gel volume. The most commonly used cross-linking agent is N,N-methylenebisacrylamide (bis-acrylamide). The polymerization reaction can be chemically initiated by using ammonium persulfate in the presence of the catalyst N,N,N,N-tetramethylethylenediamine (TEMED). The cross-linking density and pore sizes can be adjusted by selecting the concentrations of acrylamide and bis-acrylamide. For high molecular weight proteins, gels with lower densities are used, while proteins with lower molecular weights are separated on denser gels. The density of cross-linking and pore size significantly influences the migration rate, required voltage, and buffer heating. Denaturing conditions such as buffers, gels, and voltages ensure uniform physicochemical properties of protein molecules and allow for the separation of proteins based solely on differences in their masses. Polypeptides with a higher molecular weight migrate slower, while those with lower molecular weights migrate faster. Proteins have different spatial structures (secondary, tertiary, and quaternary structures) and different charges due to their amino acid composition. These parameters significantly affect the ability and speed of protein molecule migration through the polyacrylamide gel pores in the presence of an electric field. As the movement speed of proteins is a result of shape, charge, and mass, native proteins cannot be separated on a gel solely by mass. For protein separation, SDS-PAGE electrophoresis is commonly used in the presence of SDS (sodium dodecyl sulfate). SDS (sodium dodecyl sulfate) is an ionic detergent (surfactant) that significantly enhances the resolution of electrophoretic techniques. Treating a protein molecule with SDS results in the formation of protein-SDS complexes with a fixed ratio of electric charge to mass. SDS binds to the hydrophobic groups of amino acids, giving proteins a negative charge, masking the protein's original charge (Fig. 1). This causes them to migrate towards the positive anode. The amount of bound SDS is proportional to the size of the protein molecules (1g of protein binds 1.4g of SDS). As a detergent, SDS also contributes to the Strona 2 z 6  WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 denaturation of protein spatial structures, leading to the unfolding of the polypeptide chain. Additionally, staining the SDS-protein complexes is much more efficient than staining the protein alone, and the presence of SDS effectively eliminates enzymatic protein degradation during separation. Fig. 1. Protein molecule surrounded by detergent molecules. Complete denaturation of a protein by the SDS detergent is not possible if the protein contains covalent bonds such as disulfide bridges. To break these bonds, reducing agents such as 1% beta-mercaptoethanol or DTT (dithiothreitol) are used. The SDS-PAGE method (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) is an electrophoresis technique in a polyacrylamide gel under denaturing conditions in the presence of the SDS detergent. This method allows for: accurate determination of the molecular weights of separated proteins, identification and monitoring of protein mixtures' composition, checking the homogeneity of proteins, analysis of the subunit structure of biologically active protein complexes. Strona 3 z 6  WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 Practical Part Student: ✓ prepares the electrophoresis buffer, ✓ prepares protein samples, ✓ applies samples to the gel, ✓ conducts protein electrophoresis, ✓ interprets the electrophoresis results. Protein samples examined in the SDS-PAGE exercises: A) FBS (Fetal Bovine Serum) - fetal serum from calves. It is a by-product in the meat/dairy industry. FBS serum is obtained by spinning clotted blood collected from the fetal heart. To use FBS in cell cultures, the serum must undergo sterilization, filtration, and quality control. In classes, we will separate proteins present in the mentioned serum. B) BSA (Bovine Serum Albumin) - albumin derived from bovine serum. It is extensively obtained from cow blood (also cattle and similar). Commonly used in biochemistry and molecular biology as a neutral, non-reactive protein that does not disrupt most conducted reactions, and it does not exhibit enzymatic activity. When purified, it is free from antibodies (also present in serum) and other organic impurities. Some uses of BSA include: blocking the unoccupied binding surface of nitrocellulose (or other substrates) in procedures like western blot, dot blot, ELISA, and similar, as well as "saturating" the surfaces of reaction vessels to prevent reagents or contaminants from adhering to them, stabilizing enzyme functions (for example, restriction or polymerase chain reaction enzymes), stabilizing buffers used for storing enzymes, as a protein that reinforces specific interactions in reactions with antibodies, as a protein for creating standard curves, reference protein, or "blind" test protein, a nutrient in cell cultures. C) Animal-origin milk. Milk is the secretion of female mammals produced during lactation, serving as the primary source of nutrition for young individuals of the species and a component of many food products. The milk's composition is genetically and environmentally determined. Milk contains five main groups of compounds: proteins, carbohydrates, fats, minerals, vitamins. The primary protein in milk is casein (mass 100-150 million Da), which divides into several Strona 4 z 6  WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 fractions differing in mass. Milk also contains proteins from the albumin group (alpha- lactoalbumin, beta-lactoglobulin, and serum albumin) and immunoglobulins. SDS-PAGE Separation Procedure Reagents: running buffer for electrophoresis: 25 mM Tris, 192 mM glycine, 0.1% SDS, protein electrophoresis gels prepared by instructors or commercially available pre-cast polyacrylamide gels, four times concentrated Laemmli denaturing buffer for proteins: 0.9 ml Laemmli buffer + 0.1 ml beta-mercaptoethanol, bovine serum albumin (BSA), fetal bovine serum (FBS), animal-origin milk, milk of non-animal origin, protein power drink, “old” protein molecular weight marker, “new” protein molecular weight marker with bands sized: 250, 150, 100, 75, 50, 37, 25, 15, 10 kDa. Strona 5 z 6  WORKSHEET 7- MOLECULAR BIOLOGY / Academy of Silesia / Year I; sem. 1 / 2024/25 SDS-PAGE Stages: 1. Dilute the 10x electrophoresis buffer (Tris/Glycine/SDS) with double-distilled water (80 ml of concentrated buffer diluted to 800 ml ddH2O). 2. Dilute FBS, BSA, milks and protein power drink with ddH2O according to the third and fourth column of the table below, then mix with the Leammli buffer containing beta- mercaptoethanol (fifth column in the table). Mix thoroughly by pipetting several times. Prepare 40 µl samples in the following manner: Well Marker Mass/Sample Water µl Protein µl Leammli Buffer µl µl Sample per Gel 1 Marker Mass (New) 5 2 FBS 28,5 1,5 10 5 3 PCR Mix Plus Green 25 5 10 15 4 BSA(10 µg/ul) 28 2 10 5 5 20 10 10 5 6 Proteinase K 28,5 1,5 10 5 7 UHT Milk 28,5 1,5 10 5 8 Soy milk 27 3 5 9 Almond milk 20 10 10 5 10 Protein power drink 27 3 10 5 3. The prepared samples should be thermally denatured by incubating in a thermal block at 99°C for 10 minutes. 4. Prepare the gel for electrophoresis: remove the comb, remove the gel protective tape, assemble the vertical electrophoresis apparatus, check the tightness of the installation. 5. Apply the samples and marker to the wells in the gel in the amounts indicated in the table. 6. Conduct electrophoresis at 200V for approximately 40 minutes. 7. Capture gel images using the GelDoc system utilizing Bio-Rad's StainFree technology. Required literature for self-study before the class by the student: "Cell Biology Basics" by B. Alberts Chapter 4, only the first subchapter "Shape and Structure of Proteins" (I, II, III, IV, protein secondary structure, interactions in the polypeptide chain, protein charge) fifth edition pages 117-136 + protein electrophoresis panel 4-5 p. 167. The book is available in the university library. Strona 6 z 6