SDS-PAGE (Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis)

Updated 11/3/2023 07:45 AM

by RIVERA-OMICS (created by Os.)

Introduction

SDS-PAGE (Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis) is a method for separating proteins by molecular weight. Recipes for buffers can be found on the western blot recipes sheet.

Protocol Authors: Osvaldo D. Rivera, PhD & Luis Vazquez Quiñones, PhD

Materials

Protein Samples

Pippettes & Tips (P₂₀, P₂₀₀)

Microtubes

Floating Microtube Rack

Beaker

Hot Plate

Acrylamide Solution (40%)

SDS-PAGE Running Buffer 1X

Stacking Gel Buffer

4X Laemmli Sample Buffer (4X LSB)

Bio-Rad Tetra Cell System

BioRad Precision Plus Protein Dual Color

Isopropanol (2-propanol)

N,N,N′,N′-Tetramethylethylenediamine (TEMED)

Procedure

Glass Cassette and Casting Stand Assembly

Place the casting frame upright with the pressure cams in the open position and facing forward on a flat surface.

Select a spacer plate of the desired gel thickness (0.75 mm, 1.0 mm & 1.5 mm) and place a short plate on top of it.

Orient the spacer plate so that the labeling is "up". Slide the two glass plates into the casting frame, keeping the short plate facing the front of the frame (side with pressure cams).

Note: Ensure that both plates are flush on a level surface and that the label on the spacer plate is oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly.

When the glass plates are in place, engage the pressure cams to secure the glass cassette sandwich in the casting frame. Check that both plates are flush at the bottom.

Put the gray casting stand gasket (made of thermoplastic material) clean and dry. Place the casting frame into the casting stand by positioning the casting frame (with the locked pressure cams facing out) onto the casting gasket while engaging the spring-loaded lever of the casting stand onto the spacer plate.

Note: The gray casting stand gaskets must be CLEAN and DRY. The casting stand gaskets are made of a special thermoplastic material that swells when soaked in water, so we recommend that you DO NOT soak the gaskets for prolonged periods prior to casting. If the gaskets do get accidentally soaked and display swelling and/or deformation, just allow them to air dry and they will regain their original shape, size, and performance.

Gel Casting: Make a Discontinuous Polyacrylamide Gel

Place a comb completely into the assembled gel cassette. Mark the glass plate 1 cm below the comb teeth. This is the level to which the resolving gel is poured. Remove the comb.

Prepare the resolving gel monomer solution according to the recipe sheet by combining all reagents except APS and TEMED.

CRITICALAdd APS and TEMED according to the recipe sheet to the degassed monomer solution and pour to the mark using a glass or disposable plastic pipette. Pour the solution smoothly to prevent it from mixing with air.

Immediately overlay the monomer solution for the resolving gel with isopropanol (2-propanol) or 0.1% SDS with a transfer pipette slowly by the corners of the glass cassette.

Allow the resolving gel to polymerize for ~30 minutes. Remove alcohol from the gel top and rinse the gel surface completely with distilled water.

00:30:00

Prepare the stacking gel monomer solution according to Table 1. Combine all reagents except APS and TEMED.

00:15:00

Dry the top of the resolving gel with filter paper before pouring the stacking gel.

Add APS and TEMED according to the recipe sheet to the degassed stacking gel monomer solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.

Insert the desired comb according to the gel thickness between the spacers starting at the top of the spacer plate, making sure that the tabs at the ends of each comb are guided between the spacers. Seat the comb in the gel cassette by aligning the comb ridge with the top of the short plate.

Allow the stacking gel to polymerize for ~30 minutes.

00:30:00

Rinse the casting frame(s) and stand with distilled, deionized water after use.

Assembling the Mini-PROTEAN Tetra Cell Electrophoresis Module

Note: When running 2 gels only, use the electrode assembly (the one with the banana plugs), NOT the companion running module (the one without the banana plugs). When running 4 gels, both the Electrode Assembly and the Companion Running Module must be used, for a total of 4 gels (2 gels per assembly).

Set the clamping frame to the open position on a clean flat surface.

Place the first gel sandwich or gel cassette (with the short plate facing inward) onto the gel supports; gel supports are molded into the bottom of the clamping frame assembly; there are two supports in each side of the assembly. Note that the gel will now rest at a 30° angle, tilting away from the center of the clamping frame. Please use caution when placing the first gel, making sure that the clamping frame remains balanced and does not tip over. Now, place the second gel on the other side of the clamping frame, again by resting the gel onto the supports. At this point there will be two gels resting at an angle, one on either side of the clamping frame, tilting away from the center of the frame.

Note: It is critical that gel cassettes are placed into the clamping frame with the short plate facing inward. Also, the clamping frame requires 2 gels to create a functioning assembly, If an odd number of gels (1 or 3) is being run, you must use the buffer dam.

Using one hand, gently pull both gels towards each other, making sure that they rest firmly and squarely against the green gaskets that are built into the clamping frame; make certain that the short plates sit just below the notch at the top of the green gasket.

While gently squeezing the gel sandwiches or cassettes against the green gaskets with one hand (keeping constant pressure and both gels firmly held in place), slide the green arms of the clamping frame over the gels, locking them into place. Alternatively, you may choose to pick-up the entire assembly with both hands, making sure that the gels do not shift, and simultaneously sliding both arms of the clamping frame into place. The arms of the clamping frame push the short plates of each gel cassette up against the notch in the green gasket, creating a leak- proof seal (check again to make certain that the short plates sit just below the notch at the top of the green gasket). At this point, the sample wells can be washed-out with running buffer, and sample can be loaded. If running more than 2 gels, repeat steps 1a–d with the Companion Running Module.

Note: Do not attempt to lock the green arms of the clamping frame, without first ensuring that the gel cassettes are perfectly aligned and stabilized against the notches on the green gaskets of the module. To prevent the gels from shifting during the locking step, firmly and evenly grip them in place against the core of the module with one hand.

CAUTION: When running 1 or 2 gels only, DO NOT place the Companion Running Module in the tank. Doing so will cause excessive heat generation and prevent electrophoretic separation.

Place the assembly inside the mini tank chamber.

Gently remove the comb and rinse the wells thoroughly with running buffer.

Sample Loading

Fill the assembly (upper chamber) and the tank (lower chamber) with buffer. Fill the tank (lower chamber) with buffer to the indicated level (550mL for 2 gels and 680mL for 4 gels).

Load the samples into the wells with a micropipette using gel loading tips.

Load 10 μL (for immunoblot purposes or 5 μL for staining purposes) of the Precision Plus Protein Standard Dual Color on lane 1.

Load 20-40 μL of each of your protein samples per lane, depending of the gel thickness.

Note: You may use a sample loading guide to locate the sample wells. Place the pipette tip into the slots of the guide and fill the corresponding wells.

CRITICALLoad samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe needle or pipette.

Running the gel

Insert the electrical leads into a suitable power supply with the proper polarity.

Apply power to the Mini-PROTEAN Tetra cell and begin electrophoresis; 200V constant is recommended for SDS-PAGE and most native gel applications. The same voltage (200V) is used for both 2 and 4 gels. The optimal voltage for your application may differ. Run time is approximately 35 minutes (Electrophoresis time will vary between 35 and 45 minutes for Tris-HCl gels, depending on acrylamide percentage levels) at 200V for SDS-PAGE.

If your purpose at the end is to have a beautiful image you have to run the gels at 100V; use 200V when time is important.

00:01:00

Gel Removal

After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.

Remove the tank lid and carefully lift out the electrode assemblies. Pour the running buffer into an appropriate container for recycling up to 3 times or, if disposing, then dilute it with an equal volume of water and the pour into the drain with constant running water.

Note: Always pour off the buffer before opening the arms of the assembly, to avoid spilling the buffer.

Open the arms of the assembly and remove the gel cassettes.

Remove one of the gel plates by gently inserting the Bio-Rad green cutter through the space between plates in the stacking gel separating one of the plates.

The gel will stay attached to the other glass plate and with the gel cutter, cut just a little bit of the lower right corner of the gel.

a) This will help you to always visualize the gel in the proper orientation (in other words in the order that you loaded your samples).

b) Lane 1 will always be protein standard.

Remove the gel by detaching one of the corners with the gel cutter and the glass plate inverted over the plastic container with MQ dH₂O; gravity will do the rest.

Rinse the Mini-PROTEAN Tetra cell electrode assembly, clamping frame, and mini tank with distilled, deionized water after use.