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Separation of Bacterial Proteins using SDS-PAGE

Separation of Bacterial Proteins using SDS-PAGE

INTRODUCTION

Aims:
i. To apply and appreciate gel electrophoresis as a protein spoliation and purification protocol
ii. To use SDS-PAGE electrophoresis in protein purification

Background

Polyacrylamide Gel Electrophoresis (PAGE) of proteins has become an important tool for examination of bacteria in the study of their protein biochemistry at the molecular level. The SDS-PAGE is the most popular of the techniques employed for the separation of polypeptides in complex mixtures as well as for the determination of their molecular weights (Molloy, et al., 2008). Sodium dodecyl sulfate (SDS) is an anionic detergent that binds to a polypeptide in proportion to its size. When treated with SDS and a reducing agent, polypeptides become rods of negative charge, but with equal charge densities. The intrinsic charge on polypeptides is effectively overwhelmed by SDS binding thus allowing their migration and separation solely according to size (Molloy, et al., 2008). The SDS-PAGE technique separates proteins by the more conserved parameter of molecular weight and thus appears to detect broader taxonomic relationships, especially at the species and subspecies levels, than methods based on charge parameters. One-dimensional SDS-PAGE offers the combination of high-resolution and good reproducibility and thus has clear advantages over the lower resolving nondenaturing systems and the lower reproducibility of both gradient-PAGE and isoelectric focusing. The theoretical background for the SDS-PAGE technique has been described in detail elsewhere and is not considered further in this chapter.
Electrophoresis is the study of movement of charged molecules in an electric field. The generally used support medium is the cellulose or thin gels made up of either polyacrylamide or Agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acids or carbohydrates, whereas Agarose and polyacrylamide gels are widely used for larger molecules like proteins.
However, it is worth noting that the conventional electrophoresis technique cannot be used to measure the molecular weight of biological molecules because the mobility of the substance in the gel is influenced by both charge and size. Nevertheless, this problem can be solved by treating the proteins of interest so that they have a uniform charge (Molloy, et al., 2008). In this manner, electrophoretic mobility will depend only on the molecular size of the proteins. The molecular weight of proteins maybe estimated if they are subjected to electrophoresis in the presence of a detergent called sodium dodecyl sulfate and a reducing agent called mercaptoethanol (Wittig, Braun & Schägger, 2006).
The purpose of SDS is to provide with a rapid and relatively accurate way to determine proteins molecular weights. Masses determined by SDS-page are usually accurate within 5-10%, although occasionally proteins may retain enough secondary structure or contain sufficient charged groups to migrate anomalously. The migration of histones, which carry a strong intrinsic charge, is an example of this phenomenon.

Continuous and discontinuous categories of buffer systems are available for SDS-page. In the first category, continuous systems use the same buffer in both gel and tank. On the other hand, the continuous gels are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels (Wittig, Braun & Schägger, 2006).. In addition, discontinuous buffer systems employ different buffers to tank and gel, and often two different buffers within the gel, with a third buffer in the tank. Discontinuous systems concentrate, or ‘stack’ the proteins samples into a very narrow zone prior to separation, which results in improved band Sharpness and resolution.

REQUIREMENTS

Stock acrylamido solution: Acrylamido 30 %(w/v), Bis acrylamido 0.8%(w/v)- make up to 250 ml in d H2O
Buffers
i. 1.875 M Tris-HCL,Ph8.8-113.5/500 ml to adjust ph with HCL
ii. 0.6M Tris-HCL,PH 6.8-36.6G/500ml to adjust ph with HCL
Gel polymerizers- 10 %( wt/v) Ammonium persulfate(1.0 g/10 ml)
10% SDS (in water)
Electrode buffer
i. Tris 0.05 M
ii. Glycine 0.384 M
iii. SDS 0.1% (make up to 2 ltrs with water, NO PH adjustment)
Sample buffer (50 ml)
i. 0.6 M Tris-HCL, ph 6.8
ii. Sucrose 5g
iii. SDS 0.5g
iv. Mercaptoethanol 0.25 ml
v. Bromophenol blue 5.0 ml (from a 0.5 % stock)

PROCEDURE

GEL CASSETTE ASSEMBLY

A short plate was placed on top of the spacer plate. Both plates were then inserted into the green casting frame on a flat surface ensuring that the “legs” of the casting frame were down. The casting frame was then clamped making sure that the plates were level on the bottom. The cassette assembly was placed on a flat surface so the plates are level. The well forming comb was inserted into the opening between the glass plates. The reagents were then combined (except TEMED) in a small beaker in the order listed above.

ACRYLAMIDE GEL UNDER SDS-PAGE CONDITIONS

The Gilson’s pipettes were used for adding the following into 100ml flask.
Gel composition 10% 15%
Water 2.85 4.45
1.875 M Tris (ml) 2 2
30%acrylamide(ml) 3.4 5
10%SDS (µl) 100 100
10%APS (µl) 50 50
TEMED (µl) 10 10
Total volume (ml) 5 5

The content was then degassed before adding TEMED, so as to remove oxygen which inhibits polymerization step. Once the gel was ready to pour, TEMED was quickly added and then mixed by swirling gently; the solution was then drawn into 10 ml syringe then gently dispensed between the glass plates.
The solution was added till about 0.3 cm from the bottom of the placed comb was reached. The remaining acylamide solution was ejected back into the small beaker. Polymerization of the solution indicated complete polymerization of the gel between the plates. The gel was allowed to polymerize for 30 minutes.

The stacking gel was then prepared by mixing the reagents listed below in the indicated proportions:
Stacking gel composition 4%
Water(ml) 3.75
1.875 M Tris(ml)l 0.5
30% acrylamido(ml) 0.68
10% SDS(ml) 50
10% APS(ul) 25
Total Volume(ml) 5

When the separating gel had set, the overlaid water was removed. 5 ul TEMED was added to the stacking gel solution and then poured into the cassette separating the gel until the solution reached the cat-away edge of the plate. The well-forming comb was placed into the solution and left to set for 20 minutes.

The gel cassettes were then removed from the casting stand and then placed in the electrode assembly with the short plate on the inside. The buffer dam plate was placed opposite the gel cassette assembly. A slight tip ward pressure was applied on the gel cassette and the buffer dam while clamping the frame to secure the electrode assembly. The assembly was placed into the electrophoresis tank. Then, 500 ml of 1x electrophoretic buffer was prepared. The inner chamber was completely filled with 1x electrophoretic buffer so that it flooded over to fill the wells. A pipette was used to pipette the buffer into each well to remove debris.When all wells were sufficiently clean, a gel loader pipette tip was used to slowly pipette a maximum of 10 ul of sample and molecular weight marker into individual wells.
Enough electrophoretic buffers was added to the bottom of the tank to cover the bottom of the cassette assembly. The tank was covered with a lid aligning the electrodes appropriately. The electrophoresis tank was connected to the power supply of appropriate voltage, power and current applied. Once electrophoresis was complete, the power supply was turned off and the gel removed for staining.

STAINING OF PROTEINS IN GEL

50 ml of stain was poured into a plastic then the gel was immersed into coomassie solution, just enough to cover the gel. It was gently swirled for 30 minutes then the stain poured back into the container for re-use. Enough de-stain solution was poured and shaking was continued till complete de-staining was achieved.
DISCUSSION
The SDS used in protein analysis has a number of characteristics that make it necessary in protein studies. It is an aliphatic surfactant that denatures proteins by binding to the protein chain with its hydrocarbon tail, exposing normally buried regions and coating the protein chain with surfactant molecules. The polar heads group of SDS adds an additional benefits to the use of this denaturant. Proteins solubilized in SDS bind the detergent uniformly along their lengths to a level of 1.4 g SDS/g protein. This creates a charge/mass ratio which is consistent between proteins. For this reason, separation on polyacrylamide gel in the presence of SDS occurs by mass alone. It disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively charged SDS molecules. Mercapto-ethanol assists the protein denaturation by reducing all disulphide bonds.

The procedure occurs as follows:
N N methylene bis acrylamide + Acrylamide

Chemical polymerization Ammonium persulfate (catalyst) +TEMED

Polyacrylamide

During electrophoresis, glycine enters the stacking gel, where equilibrium favours the zwitterionic for with zero charge. The glycine front moves slowly through the stacking gel, lagging behind thestrongly charged smaller Cl ions. As this two current carrying species separate, a region of low conductivity, with a consequent high voltage drop, is created between them. This zone sweeps the proteins rapidly through the large pores of the stacking gel, collecting the sample and depositing it at the top of the resolving gel in a focused narrow band.
Dyes or metals are important in staining procedure. Each different technique has its own characteristics and limitations with regard to the sensitivity and type of protein stained best (Wittig, Braun & Schägger, 2006).. All stains interact differently with the proteins in a gel in proportion to the mass of the protein. The standard coomassie brilliant blue G-250 is probably the best staining technique but less sensitive as it detects only about 0.3ng/band. It involves fixation of the protein in the gel matrix. Use of this stain is not easily reversible, and most often the protein cannot be recovered intact for other procedures.

CONCLUSION

Polyacrylamide Gel Electrophoresis (PAGE) of proteins has become an important tool for examination of bacteria in the study of their protein biochemistry at the molecular level. The SDS-PAGE is the most popular of the techniques employed for the separation of polypeptides in complex mixtures as well as for the determination of their molecular weights. Protein analysis is made easy with SDS-PAGE by ensuring that they have a uniform charge. In this manner, electrophoretic mobility will depend only on the molecular size of the proteins.
 
References
Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J. C., … & Gooley, A. A. (2008). Extraction of membrane proteins by differential solubilization for separation using two‐dimensional gel electrophoresis. Electrophoresis, 19(5), 837-844.
Schägger, H., & von Jagow, G. (2001). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical biochemistry, 199(2), 223-231.
Wittig, I., Braun, H. P., & Schägger, H. (2006). Blue native PAGE. Nature protocols, 1(1), 418-428.

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