GTB 204 Molecular Biology Protocols 2001

SDS-PAGE Principle
Electrophoresis is the study of the movement of charged molecules in an electric field. The generally used support medium is 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 acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins. The general electrophoresis techniques cannot be used to measure the molecular weight of the biological molecules because the mobility of a substance in the gel is influenced by both charge and size. In order to overcome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size. The molecular weight of protein maybe estimated if they are subjected to electrophoresis in the presence of a detergent sodium dodecyl sulfate (SDS) and a reducing agent mercaptoethanol (β ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with negatively charged SDS molecules. 1.4grams of SDS binds per gram of protein.

Mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross linking agent N N’ methylene bis acrylamide Acrylamide + N N’ methylene bis acrylamide

Chemical Polymerization

Ammonium persulfate (catalyst) +
TEMED (N,N N’ N’ tetramethylethylene diamine)

Polyacrylamide

Procedure: 1. Assembling the glass plate (Demonstration)
Note: 1. Gloves should be worn at all times while performing SDS-PAGE. 2. To insure proper alignment and casting, the glass plates, spacers, combs and casting stand gaskets must be clean and dry. The glass plates should be cleaned with 70% ethanol. 1. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then place 2 spacers of equal thickness along the rectangular plate. Next place the shorter glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are aligned (Figure 1). Loosen the 4 screws on the clamp assembly and stand it up so that the screws are facing away from you. Firmly grasp the glass plate sandwich with the longer plate facing away from you, and gently slide it into the clamp assembly. Tighten the top 2 screws of the clamp assembly. Place the clamp assembly into the alignment slot of the casting stand so that the clamp screws face away from you. Loosen the top 2 screws to allow the plates and spacers to sit firmly against the casting stand base. Gently tighten all the screws.

2.

3.

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6. 6. -3. so that the glass plates rest on the rubber gasket. Combine all reagents except APS and TEMED. Pour the water overlaying the gel and drain the excess water with strips of filter paper. 7. Add APS and TEMED to the monomer solution and mix well by swirling gently. place the clamp assembly on the other side of the alignment slot. If not. 2. Place a comb in the gel sandwich. With a marker pen. Use a steady. Figure 1 5. Prepare the separating gel monomer solution b combining all reagents except ammonium persulfate y (APS) and TEMED. This will be the level to which the separating gel is poured. Make sure that they are aligned. Check that the plates and spacers are aligned. Please refer to appendix 1 for the recipe. Deaerate and mix the solution by swirling gently.GTB 204 Molecular Biology Protocols 2001 4. Immediately overlay the monomer solution with 1 ml. Prepare the stacking gel monomer solution. Pipette the solution to the mark. 4. Do this by inverting the gel sandwich and looking at the surface of the 2 glass plates and the spacer. even rate of delivery to prevent mixing with the gel. of water. Place a comb completely into the assembled gel sandwich. Casting the gels (Demonstration) Prepare 10% resolving/separating gel and 4. Snap the acrylic plate underneath the overhang of the casting slot. realign the sandwich as in steps 1 Before transferring the clamp assembly to the casting slot. Remove the comb. Deaerate and mix the solution after adding each reagent by swirling the container gently. 2 . If 2 gels are to be prepared. Transfer the clamp assembly to one of the casting slots in the casting stand. 1. Press the acrylic pressure plate bottom. place a mark on the glass plate 1 cm below the teeth of the comb. Allow the gel to polymerize for 45 minutes to 1 hour. 2. 3. Pull the completed sandwich from the alignment slot. 5.5% stacking gel. Do not push the glass plates or spacers because this could break the glass plate. recheck the alignment of the spacers.

2. Do not pipette the pellet at the bottom of the microfuge tube. Wt Content Marker Tube 2 Vector protein Tube 3 Uninduced clone protein Tube 4 Induced clone protein induced using IPTG for next four hours. the recombinant protein is produced as follows The clone was grown for 4hours and resuspended in PBS Each group is provided with the following Procedure Tube 1 Mol. Gel is placed in the buffer chamber and running gel buffer is added into the chamber 9. Allow the gel to polymerize for 15 minutes. SDS. Rinse the syringe a few times with distilled water after loading. Preparation of samples (By students) From the recombinant clone VC-25. Insert the syringe to about 1 mm from the well bottom -2 before delivery. Rinse the syringe to be used for loading samples a few times with distilled water. Mercaptoethanol. 11. The culture was pelleted and Sample buffer (Glycerol. Loading the samples (By students) 1. 10. Bromophenol blue dye) - 20µl 20µl 20µl Boil for 5 min Do not boil Boil for 5 min Boil for 5 min Boil for 5 min Brief spin Loading sample into the well Not required 10µl 5 min 20µl 5 min 20µl 5 min 20µl 4. 3 . Remove the comb. Rinse the syringe a few times with distilled water after loading. 3. Demonstrators will load the first well with LMW (7 µl of LMW).GTB 204 Molecular Biology Protocols 2001 8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely. Load the second and other well with 20 µl of VC-25 protein as described above.

4. 3. Attach the electrical leads to a suitable power pack with the proper polarity (black to black and red to red). 7.8 Distilled water Ammonium persulfate TEMED 0. Destain the gel in a destaining solution a few times until protein bands are visualised.65 ml 1.8% Bis-acrylamide 4X Tris-HCl/SDS. agitate it slowly on a shaker. 30% acrylamide + 0.5% stacking gel 30% Acrylamide + 0. Check that the buffer in the upper buffer chamber are full because leakage of the buffer may occur.56 ml 25 µl 10 µl Preparation of a 4. Stain the gel for 1 hour. Make sure that the connection is correct.GTB 204 Molecular Biology Protocols 2001 5. Gently twist the spacer so that the upper glass plate pulls away from the gel. 6. ie. Place the lid on top of the lower buffer chamber. Make sure that the gel is fully submerged in the staining solution. Cut the gel on one side (to orientate the gel). 6.05 ml 25 µl 5 µl 4 . Push one of the spacers out to the side of the plates without removing it. Remove the gel from the buffer chamber Loosen all four screws of the clamp assembly and remove the glass plate sandwich from it. Appendix 1 Preparation of a 10% Resolving/separating gel.25 ml 3. pH 8. Running the gel 1. Approximately determine the molecular weight of the visualised protein bands by comparing them with the molecular weight markers. black to black and red to red. pH 6.8 Distilled water 10% ammonium persulfate (APS) TEMED 1. Removing and staining the gel (By tutor) 1. 4. 9. Stop the electrophoresis when the tracker dye is ~ 1 cm above the end of the glass plates. 3. 2.25 ml 0. 5. Remove the gel by gently grasping two corners of the gel and place it in the container containing the Coomassie blue stain. 2. Run the gel at a constant current of 30 mA.94 ml 1.8% Bis-acrylamide 4X Tris-HCl/SDS. 8.

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