SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel [PDF]

SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette and casting stand Put the short plate in front of the spacer (tall) plate and put in the casting frame. Orient the spacer plate so that labeling is “up”. Lock the casting frame and make sure that the bottoms of both plates are flat. Place this casting frame in casting stand and lock it. To check whether the apparatus is leaky or not, add a little bit of water to the glass plates. In the case of no leaks, dry the water between the glass plates with a piece of paper. 2. Gel preparation and casting Place the comb between the glass plates and make a mark on the glass plate 1cm below the comb teeth. Moreover, this mark is helpful to know the label of poured resolving gel. Remove the comb. Prepare the 15% resolving gel monomer solution (10 ml) using following reagents except 10% APS10 and TEMED15. 2.4ml dH2O 5.0ml Acrylamide/Bis20 2.5ml Resolving buffer25 0.1ml 10% SDS30 Vortex thoroughly and open the cap and degas for 15 min under vacuum. Finally add 50µL 10% APS and then 5µL TEMED in fume hood and the resolving gel is ready to be cast. TEMED is going to be added in the last because it starts the polymerize reaction Pour the resolving gel in between the glass plates and all the way to the marked line. At this point, air bubbles created unintentionally doesn’t matter. Finally add a layer of 15% isopropanol35 (fill all the way to the top) to remove the air bubbles created. Allow the resolving gel to polymerize for 40-50 minutes. Check whether unused resolving gel solidifies. Now prepare the 5% stacking gel monomer solution (10 ml) using following reagents except 10% APS10 and TEMED15. 5.7ml dH2O 1.7ml Acrylamide/Bis20 2.5ml stacking buffer40 0.1ml of 10% SDS30 Vortex thoroughly and open the cap and let it degas for 15 min under vacuum. Finally add 50µL of 10% APS and then 10µL TEMED and the gel is ready to cast. TEMED is added at the end because it initiate the polymerize reaction.

Get rid of the 15 % isopropanol35 using filter paper and dry the top of resolving gel before pouring the stacking gel. Pour the stacking gel in between the glass plates until the top of short plate is reached. Insert the comb carefully in space between glass plates and allow the stacking gel to polymerize from 30-40 minutes. Gently remove the comb and rinse all the wells with dH2O. Here gel cassette is ready to go.

3. Electrophoresis Module assembly Place the gel cassette onto the gel supports such that the short plate faces inward. Place the second gel cassette similarly on the other side or use dummy plastic plate (buffer dam) and lock it. Put this entire assembly in green gasket making sure that the short plates sit just below the notch at the top of green gasket. 4. Filling the Assembly with 1x electrode running buffer (running buffer) 45 Two chambers are formed in gasket, inner chamber between the two gel cassettes and two outer chambers between gel cassette and green gasket. Fill the inner chamber with electrode running buffer to the top of short plate such that wells can be washed out with running buffer. Now fill the running buffer in the outer chamber to just under the edge of outer gel plate (see marks on gasket for 1 gel or 2 gels). 5. Sample Preparation and loading Gel all the collected samples of protein (20-50 µl) and name it properly. Add 25 µl of βmercaptoethanol50 to 475 µl sample buffer (Laemmli or SDS reducing buffer, Blue color)55 prior to use. Add same amount (20-50 µl) of the sample buffer in all the protein samples (done under fume hood). Using the Dry Block Heater60, cook the protein samples to 90°C for 10 minutes. In this way proteins are denatured in sample buffer containing SDS and reducing agent β-mercaptoethanol. The resultant denatured polypeptides take on a rod-like shape and a uniform charge-to-mass ratio proportional to their molecular weight. Use a thermometer to keep track of the temperature. After 10minutes, take out the cooked protein samples and load all the protein samples into the wells using gel loading tips. Add the protein Marker65 into 1st or 2nd well for reference. 6. Power Conditions To apply the power to the entire assembly, connect red to red and black to black cable. From top edge of the short plate to the interface between resolving and stacking gel, set voltage 90 volts and start run. To make sure the gel is running properly, check whether the bubbles appear in the running gel. Once gel crosses the interface between resolving gel and stacking gel change the voltage to 120-150 volts. Since the Blue dye (Laemmli sample buffer mixed in protein samples) is a small molecule and moves ahead of the protein. Therefore the position of blue dye is good indicator of the protein position. Electrophoresis is complete once blue colored protein samples reach the bottom of the gel. 7. Gel removal Turn off the power supply, remove the tank lid and discard the running buffer. Remove the gel cassette from the assembly and then gently separate the glass plates for the gel.

8. Staining and Destaining of the gel Soaked the gel in the staining solution of Coomassie Brilliant Blue R-250 and rock it for 20-35 minute. The stain attaches to the protein only. Excess stain can be eluted by soaking the gel in destaining solution coomassie R-250. Then Overnight rocking will destain everything except protein. 10

10% APS (fresh daily)-Dissolve 100 mg ammonium per sulfate in 1 ml of dH2O. Make aliquots of 50 µl and store at -200C in Physics.

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TEMED - Store at room temperature in Biology.

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Acrylamide/Bis- Store at room temperature in Biology.

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Resolving buffer (500ml) – (1.5M Tris-HCl, pH 8.8) - Dissolve 90.855g of Tris Base in 400mL dH2O. Adjust pH 8.8 with 6N HCl. Bring total volume to 500mL with dH2O and store at 4°C in Physics.

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10% SDS - ?? Store at room temperature in biology.

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15% Isopropanol – Store at room temperature in Physics and Biology.

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Stacking Buffer (500 ml)- (0.5M Tris-HCl, pH 6.8) - Dissolve 30.285g of Tris Base in 400mL dH2O. Adjust pH 6.8 with 6N HCl. Bring total volume to 500mL with dH2O and store at 4°C in Physics.

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10x Electrode running buffer (1L)

Tris Base – 30.3g Glycine – 144g SDS – 10g Dissolve and bring total volume upto 1L with dH2O and store at 4°C in Physics. For use, dilute to 1x with dH2O (i.e. 100mL of 10X stock + 900mL dH2O) 50

β-mercaptoethanol - Store in Physics at 40C.

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Sample buffer (Laemmli) – Store in Biology.

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VWR® Analog Dry Block Heater - Low range knob adjusts from ambient to 100°C and high range knob adjusts from 75 to 150°C. 65 Protein Marker –“Bio-RAD Precision plus protein all Blue Standards Catalog# 161-0373”store at -200C in Physics.