Two-Dimensional Electrophoresis Sample Preparation with Amicon® Ultra Centrifugal Filter

Two-dimensional electrophoresis (2DE) is one of the most commonly used methods in proteome analysis. Briefly, proteins are separated by their isoelectric point (first dimension separation) followed by SDS-PAGE separation by molecular weight (second dimension separation). The salts and ionic detergents routinely used in sample preparation are not compatible with 2DE; the combination of high salt concentration and low protein content interferes with isoelectric focusing. One way to concentrate proteins is by acetone or TCA precipitation. However, this method has two key disadvantages:

  • Some proteins become insoluble and cannot be resolubilized in IPG buffer
  • Many salts become insoluble in acetone and precipitate along with the proteins

Method

Forty grams of bovine liver were blended in an industrial blender in 50 mL 50 mM Tris-HCl, 150 mM NaCl, containing protease inhibitor cocktail. The lysate was cleared by centrifugation, aliquoted, and stored at -20 °C. Protein concentration of 5 mg/μL was determined by BCA protein assay using bovine serum albumin standard.

For 2-DE analysis of the neat lysate, 150 μL of the extract was mixed with 50 μL IPG rehydration buffer and used to rehydrate an IPG strip. For 2-DE analysis of lyophilized lysate, the dried sample was resolubilized in 200 μL of IPG rehydration buffer, cleared by centrifugation, and used to rehydrate an IPG strip.

For 2-DE analysis of acetone-precipitated proteins, 150 μL liver lysate was mixed with 4 volumes of cold acetone and incubated for 1 h at -20 °C, followed by centrifugation at 12,000 rpm for 10 min. The supernatant fluid was decanted and the pellet was resuspended in 200 μL of rehydration buffer.

For 2-DE analysis of liver lysate prepared by ultrafiltration, 1 mL lysate was dispensed into an Amicon® Ultra-4 10,000 MWCO centrifugal device. The device was spun at 3000 x g for 40 min until approximately 100 μL of concentrated volume was remaining in the device. Nine-hundred microliters of 8 M urea, 2% CHAPS solution was added to the device, and centrifugation was repeated for 40 min. The concentrated proteins were immediately transferred to a microcentrifuge tube and the volume was adjusted to 1 mL with IPG rehydration buffer (8 M urea, 2% CHAPS, 0.002% bromophenol blue, 40 mM DTT, and 0.5% Pharmalyte). One-hundred fifty microliters of reconstituted ultrafiltrate were used to rehydrate an IPG strip in 200 μL total volume.

Results

Bovine liver lysate was prepared by homogenization in isotonic buffer with protease inhibitors. The protein concentration in the extract was determined to be 5 mg/mL. In order to successfully analyze the sample by 2-DE, it was estimated that approximately 0.75 mg of total protein was required, and thus 150 mL of the lysate was prepared in 200 mL volume by addition of 50 mL IPG rehydration buffer (20% of total volume).

2-DE of neat lysate resulted in poor separation and resolution, opresumably because of the high salt concentration and low concentration of chaotropic agents (Figure 1A). To achieve better separation, various methods of sample concentration were attempted. First, the sample was lyophilized and resuspended in IPG rehydration buffer. The resulting 2-D gel was streaky with no well-defined spots and with protein retained at both ends of the IPG strip (Figure 1B).

Acetone precipitation is another method for protein concentration. Figure 1C shows a 2-D gel of acetone-precipitated liver lysate. Because the salt content was greatly reduced compared to Figures 1A and 1B, a satisfactory separation was achieved. While protein recovery appeared to be complete, evaluating this assumption required an alternative method for protein concentration and desalting/ Ultrafiltration (UF) is a well-known method for protein concentration and desalting. Bovine liver proteins were concentrated by centrifugal UF. In this process, proteins are retained and thereby concentrated, while the salt concentration does not change and is the same in the retentate and the ultrafiltrate. However, because of the reduced volume of the retentate, the salt-to-protein ratio in the retentate is lower, and the sample is easily rendered suitable for 2-DE after dilution with IPG buffer.

Figure 1D shows a 2-D gel of liver proteins prepared by UF. As expected, the separation was successful, resulting in multiple well-defined spots on the 2-D gel.

Figure 1A

Figure 1B

Figure 1C

Figure 1D

 

Figure 1. 2-DE gel of 0.75 mg (in 200 μL total volume) bovine liver lysate prepared (A) by mixing proteins with IPG rehydration buffer (B); by lyophilization and resuspension in IPG rehydration buffer (C); by acetone precipitation (C), and (D) by UF. Results show that using ultrafiltration to concentrate a 2-DE lysate sample yields better resolution and higher protein recovery without detectable loss of low-molecular-weight species.

 

 References

  1. Chernokalskaya et al. Ultrafiltration for proteomic sample preparation. Electrophoresis 2004, 25, 2461–2468.