The overall goal of this novel separations technique is to fractionate native samples with high recovery and resolution while maintaining non-covalent macromolecular interactions such as protein complexes. This method can help answer key questions in the study of native macromolecular complexes, such as complex composition and stoichiometry. The main advantage of this technique is its high recovery and low sample dilution, unrivaled by any other native separations method.
Begin with assembling the casting system. The first step is to use a hot blade to cut the tip off a 20 milliliter serological pipette. Cut orthogonally 10 centimeters from the top of the dispensing tip.
Then, clamp the cut pipette tip down to a support stand. Next, cut a three centimeter length of tubing to attach to the tip. Stretch one end of the tubing over the tip, and attach a flow control valve.
Connect the valve to the dispensing tip of a gradient mixer using another piece of tubing. Keep the valve open for now. Next, put the gradient mixer on a magnetic stirrer and load stir bars into the mixing chambers.
Now, text the system for leaks using de-ionized water. Then, dry the system by blowing air through it. Cover the top of the pipette section with two layers of tape.
Then, puncture a hole through the tape layers, and slide a glass tube through the hole. Extend the glass tube to within two millimeters of the bottom. The tube must fit the tape snugly and not touch the pipette walls.
Now, cast the gel. Close the dispensing valve, and add the 12%T separating gel solution to the mixing chamber furthest from the dispensing tip. So bubbles don't form, open the valve between the chambers to let the solution flow.
Then, close the valve and transfer the dislocated gel solution back to the previous chamber using a pipette. Now, add 1%T gel to the mixing chamber closest to the dispensing tip, and turn on the magnetic stirrer. Now, open the flow valve, then both gradient mixer valves at the same time to allow the two gel solutions to mix and flow into the glass tube.
When finished, attach the tubing to a 10 milliliter syringe, keeping it above the liquid level. Then, use the syringe to push the remaining gel solution from the tubing into the pipette and tube. Before any air gets in, close the valve.
Next, gently pipette about 0.25 milliliters of water saturated butanol over the gel to seal the polymerization reaction. Let the reaction go overnight at room temperature. On the following day, carefully peel the tape off the top of the device, disconnect the valve from the tubing under the pipette, and release the pipette from the clamp.
Over a sink, attach a 10 milliliter syringe filled with de-ionized water to the tubing, and use this water to slowly force the polymerized gel out of the pipette. Then, trim the excess gel so it is smooth and flush with the glass edge. Store the gel tube in 0.1 X buffer solution, and cover it with parafilm wrap.
After manufacturing the parts for the CN-GELFrEE device, assemble them. First, fit the end of the gel tube without gel through a spacer, an O ring, and a second spacer in a sandwich fashion. Then, add the cathode buffer chamber after the second spacer.
Pass the screws fully through the side holes and tighten the nuts to both sides. Make certain that the top end of the glass tube is fixed before the top aperture of the buffer chamber and not directly under it or past it. Proceed by fitting the bottom end of the gel tube through a spacer, an O ring, the collection chamber, a second spacer, and then attach the anode buffer chamber like the cathode buffer chamber.
Make sure that the bottom end of the gel tube is fixed past the top aperture of the buffer chamber. It should also be close to the back wall without touching it. Now, clamp the electrophoresis device vertically, with the cathode up.
Then, load the two chambers with either four degree Celsius anode or cathode buffer. Tap out any air bubbles, and check for leaks. The platinum electrode must touch the buffers, and both gel tube ends must be immersed in buffer.
The last step is to connect the gel to the power supply. Load the sample on the top surface of the gel using the load tip. Next, run the gel at one watt until the red dye front has passed to the bottom of the gel.
Then, firstly, turn off the power supply, and secondly, discard the buffers. Now, disassemble the bottom spacer and chamber, keeping one spacer and the O ring attached. Put the bottom end of the gel tube into the collection chamber before the top aperture but not past it or directly under it, and push the spacer and O ring close to the collection chamber.
Then, cover the aperture with a membrane rehydrated in anode buffer. Assemble the anode buffer chamber after the second spacer. Lay the device horizontally over an ice bucket.
Then, refill the buffer chambers, check for leaks, and then add 150 microliters of anode buffer into the collection chamber. Now, send a constant three milliamps to the device. Once the dye front dilutes, turn off the power.
Then, collect the first fraction. Transfer it to a low protein binding microcentrifuge tube, and put it on ice. Next, refill the collection chamber with another 150 microliters of anode buffer, and collect the next fraction.
Carefully proceed by repeating this process to collect all the fractions. With practice, no protein loss should occur. Every 60 minutes during the fractionation, change the anode and cathode buffer.
Proteins were extracted from frozen mouse heart and fractionated natively using CN-GELFrEE in a one to 12 percent T gel tube. Fraction aliquots were run on a CN-PAGE slab gel and silver stained. Each native fraction was subsequently reduced and denatured.
Samples were run again on an SDS-PAGE slab gel. Mass shifts and an increase in the number of species were observed in all fractions compared to the native fractions, indicating that the native fraction contained intact protein complexes. The native fractions were then cleaned and analyzed with mass spectrometry.
One intact protein complex observed, for example, was homodimeric malate dehydrogenase. It had a charge distribution between 16 plus and 20 plus, and a molecular mass of 72, 843 daltons. The 18 plus charge state was isolated and collisionally activated, resulting in the ejection of monomeric malate dehydrogenase with a molecular mass of 36, 421 daltons.
Source activation was applied to the intact complex in order to induce monomer ejection. This allowed isolation, further fragmentation, and identification of the monomers. Thus, CN-GELFrEE is compatible with mass spectrometry and it does not disrupt non-covalent protein-protein interactions of macromolecular assemblies.
This procedure generates liquid entrapped native fractions that are likely to be compatible with many downstream analytical methods besides mass spectrometry, including activity assays, binding assays, and microscopy. Don't forget that working with liquid fractions and electricity can be extremely hazardous, and that the electric source should always be off during the handling of liquids or of the device throughout this procedure.
