SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a method for qualitative protein analysis. It is widely used in molecular biology and protein research to separate oligonucleotides and proteins.

J&K Scientific provides SDS-PAGE related reagents, pipettes, and consumables.

Experimental Steps

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Experimental Steps

I. Preparation of SDS-PAGE Gel

1. Wash the electrophoresis glass plates with water, then wipe with alcohol and allow to air dry. Assemble the glass plates according to the instructions, and use deionized water to check whether the three edges of the glass plates leak. If leakage occurs, reassemble the glass plates and test again.

2. Prepare the separating gel as shown in the table. Add TEMED last, then quickly rotate the mixture in one direction (to prevent bubble formation). Use a 1000 μL micropipette to inject the gel into the gap between the two glass plates (fill to the upper edge of the red plate). Carefully overlay a layer of isopropanol on the gel surface to seal it.

3. Allow the separating gel to polymerize at room temperature for approximately 40 minutes. Pour off the isopropanol overlay and rinse the gel surface 3–4 times with deionized water to remove unpolymerized acrylamide. Discard the deionized water and absorb any residual moisture with absorbent paper.

4. Prepare the stacking gel as shown in the table. Add TEMED last and quickly mix by rotating. Slowly fill the gap between the glass plates completely, insert the sample comb (the side marked "1 cm" facing inward; insert one side first, then press from one side to the other to expel air bubbles), and place vertically at room temperature.

5. Polymerization is complete after approximately 30 minutes. Pull out the comb parallelly, remove the glass plates, and tear off the rubber strips sealing the glass plate clamps. Place the "concave" side of the glass plate assembly tightly against the electrophoresis tank and secure with clamps.

II. SDS-PAGE Electrophoresis

1. Add 300 mL of electrophoresis buffer to the electrophoresis tank. Sequentially add 10 μL of each of the following samples into the loading wells from the left side: pre-induction sample (without DTT), pre-induction sample (with DTT), post-induction sample (without DTT), post-induction sample (with DTT), and 5 μL of protein marker. Add an appropriate amount of 4× protein loading buffer to the remaining empty wells.

2. Close the lid and connect the power supply (red to red, black to black). Slowly adjust the voltage to 80 V and begin electrophoresis. When the bromophenol blue dye front reaches the separating gel, increase the voltage to 150 V. Stop electrophoresis when the blue dye front migrates to the bottom edge of the gel.

III. Coomassie Brilliant Blue Staining and Destaining

1. Remove the glass plates from the electrophoresis tank, pry them open, and take out the gel. Remove the stacking gel and cut off a corner from the upper right side to mark the orientation/order.

2. Place the gel in a Petri dish, add approximately 40 mL of staining solution, and stain on a decolorizing shaker for 50 minutes.

3. After staining, recover the staining solution. Rinse the gel 2–3 times with water, then add destaining solution and destain until clear protein bands are visible (typically 1–2 days).

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