Tuesday, August 25, 2009

SDS PAGE





Principle
Electrophoresis is the study of the movement of charged molecules in an electric field. The generally usedsupport 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 biologicalmolecules because the mobility of a substance in the gel is influenced by both charge and size. In order toovercome this, if the biological samples are treated so that they have a uniform charge, electrophoretic mobilitythen 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 (b ME). SDS disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptidechain 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
Add Chemical Ammonium persulfate (catalyst)
+
Polymerisation ↓ TEMED (N,N N’ N’ tetramethylethylene diamine
Polyacrylamide
Procedure:
1. Assembling the glass plate
( Gloves should be worn at all times while performing SDS-PAGE. 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 two 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
2. 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.
3. Place the clamp assembly into the alignment slot of the casting stand so that the clamp screws faceaway 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
4. Pull the completed sandwich from the alignment slot. Check that the plates and spacers are aligned. If not, realign the sandwich as in steps 1-3. Before transferring the clamp assembly to the casting slot,recheck the alignment of the spacers. Do this by inverting the gel sandwich and looking at the surface of the 2 glass plates and the spacer. Make sure that they are aligned.
5. Transfer the clamp assembly to one of the casting slots in the casting stand. If 2 gels are to be prepared, place the clamp assembly on the other side of the alignment slot.
6. Press the acrylic pressure plate bottom, so that the glass plates rest on the rubber gasket. Snap the acrylic plate underneath the overhang of the casting slot. Do not push the glass plates or spacers because this could break the glass plate.
2. Casting the gels (Demonstration)
Prepare 10% resolving/separating gel and 4.5% stacking gel.
1. Prepare the separating gel monomer solution by combining all reagents except ammonium persulfate(APS) and TEMED. Deaerate and mix the solution after adding each reagent by swirling the container
gently.
2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the Glass plate 1 cm below the teeth of the comb. This will be the level to which the separating gel is poured. Remove the comb.
3. Add APS and TEMED to the monomer solution and mix well by swirling gently. Pipette the solution to the mark.
4. Immediately overlay the monomer solution with 1 ml. of water. Use a steady, even rate of delivery to prevent mixing with the gel.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Pour the water overlaying the gel and drain the excess water with strips of filter paper.
6. Prepare the stacking gel monomer solution. Combine all reagents except APS and TEMED. Deaerate and mix the solution by swirling gently.
7. Place a comb in the gel sandwich.
8. Add APS and TEMED to the solution and pipette the solution down one of the spacer until the sandwich is filled completely
9. Allow the gel to polymerize for 15 minutes.
10. Remove the comb.
11. Gel is placed in the buffer chamber and running gel buffer is added into the chamber
3. Preparation of samples. ( here a cloned protein is put under analysis)
From the recombinant clone VC-25, the recombinant protein is produced as follows:
The clone was grown for 4hours and induced using IPTG for next four hours. The culture was pelleted and resuspended in PBS
4. Loading the samples
1. Rinse the syringe to be used for loading samples a few times with distilled water. Demonstrators will load the first well with LMW (7 ml of LMW). Insert the syringe to about 1-2 mm from the well bottom before delivery. Rinse the syringe a few times with distilled water after loading.
2. Load the second and other well with 20 ml of VC-25 protein as described above. Do not pipette the pellet at the bottom of the microfuge tube. Rinse the syringe a few times with distilled water after loading.
5. Running the gel
1. Check that the buffer in the upper buffer chamber are full because leakage of the buffer may occur.
2. Place the lid on top of the lower buffer chamber. Make sure that the connection is correct, ie. black to black and red to red.
3. Attach the electrical leads to a suitable power pack with the proper polarity (black to black and red tored). Run the gel at a constant current of 30 mA.
4. Stop the electrophoresis when the tracker dye is ~ 1 cm above the end of the glass plates.
6. Removing and staining the gel.
1. Remove the gel from the buffer chamber
2. Loosen all four screws of the clamp assembly and remove the glass plate sandwich from it.
3. Push one of the spacers out to the side of the plates without removing it.
4. Gently twist the spacer so that the upper glass plate pulls away from the gel.
5. Cut the gel on one side (to orientate the gel).
6. Remove the gel by gently grasping two corners of the gel and place it in the container containing the Coomassie blue stain. Make sure that the gel is fully submerged in the staining solution.
7. Stain the gel for 1 hour, agitate it slowly on a shaker.
8. Destain the gel in a destaining solution a few times until protein bands are visualised.
9. Approximately determine the molecular weight of the visualised protein bands by comparing them with the molecular weight markers.

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1 comments:

roshan on August 23, 2008 at 1:25 AM said...

u explained the technique very beautifully i want to add afew points to it

1. after the gel has been completely developed u can subject it to either a absorption spectra or a densitometer

2. this will help u to understand & determine many parameter of ur study


3. u can also subject this to 2d electrophoresis which my friend anand will post some day a very powerful electrophoresis technique combining SDS PAGE & iso electric focusing

my best wishes r with u all

best of luck for this wonderful website

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