Influence of Hydrogen Ion Concentrations on the Protein Mobility in Native-PAGE Buffers

Abstract

The study demonstrates the effect of hydrogen ion concentrations (pH) of Tank Buffer (TB) and resolving gel buffer (RGB) in a native-PAGE system on protein samples. A pH range of 7.8 to 9.3 for RGB and 8.3 to 9.3 for TB were used. Proteins in the samples under native-PAGE at varying pH of RGB such as 7.8, 8.3 and 8.8 with pH of TB, 8.3 and 8.8 were resolved well. The total running time for the samples to reach the end of the dye front ranged between 2.30 h to 4.30 h at the above pH combinations. It was observed as 3.30 h as total running time under normal pH conditions (RGB, pH 8.8; TB, pH 8.3). At the same time, buffers at higher pH or the combination of extreme pH (7.8 vs. 9.3) in the buffers were not favored good protein mobility/resolution, and bands were diffused. Longer running time was observed in various combinations of pH of RGB and pH of TB with 18.00 h being a longest with a pH of 8.3 and 9.3 for RGB and TB, respectively. This indicated the importance of pH of electrophoresis buffers for ionization of various proteins for better separation.

Keywords: Native-PAGE; pH; Protein Mobility; Resolving Gel Buffer; Tank Buffer

Abbreviations

TB: Tank Buffer; RGB: Resolving Gel Buffer; PAGE: Polyacrylamide Gel Electrophoresis; pIs: Isoelectric Points; BSA: Bovine Serum Albumin; RBC: Red Blood Cells; PTU: Phenylthiourea

Introduction

Acrylamide gel electrophoresis is a standard technique used to separate, analyse and purify molecules such as proteins based on their net charge and size [1-3]. Separation is influenced by the type of proteins, pH of the buffers used, electrical field strength and temperature [4,5]. The pH of the electrophoresis buffer required to be in the range over which proteins of interest are stable or at which they retain their biological activity [6]. The pH should be appropriately chosen with respect to the isoelectric points (pIs) of proteins. The pH of the resolving gel buffer must be adequately distant away from the pIs of the proteins and so they carry enough net charge to travel through the gel in a reasonable time period [7]. The resolution and stability of proteins are often decided by the choice of pH [8]. A protein possesses charge as a result of the constituents of its acidic (glutamic and aspartic acids) and basic (lysine and arginine) amino acids. However, it is only the net charge on the molecule that is significant in deciding its movement on a matrix. Since each molecule possesses unique charge and mass, it migrates to a specific position within the electric field in a given duration of time [9]. Therefore, if a mixture of proteins is subjected to electrophoresis, each of the protein would be expected to concentrate into a tight migrating band at a unique position in the electric field on a matrix [10]. Different protein molecules in the sample move through the matrix at various velocities under the influence of an applied electrical current [11]. Polyacrylamide is the most common matrix used for separating proteins [1,12]. Although studies on standardization of PAGE according to the different model proteins in regard to various pH have reported, there is no information pertaining to the effect of changes of hydrogen ion concentration in resolving gel buffer which is crucial for separation of biological macromolecules [10,13,14]. In that context, this study is aimed to optimize a polyacrylamide gel to separate native proteins with good resolution by varying the pH of buffers used in both electrophoresis and resolving gel.

Materials and Methods

Protein Samples

Lysozyme and Bovine Serum Albumin (BSA) fraction-V were purchased from Hi-Media, haemoglobin from ox red blood cells (RBC) and haemolymph of Bombyx mori (fifth instar larva) were used for this study. The average molecular weight of lysozyme is 14.6 kDa and its pI value is 11.0. The molecular weight of BSA is 66.3 kDa and pI value is 4.9. The protein concentration of samples was measured using bovine serum albumin as standard by following Lowry et al. [15]. Alsever’s solution was prepared by following Garvey et al. [16] for maintaining ox RBC.

Preparation of Protein Samples

Lysozyme muramidase from chicken egg white

Lysozyme (5 mg lyophilized powder) was weighed and dissolved in 1 ml of double distilled water (stock). From stock, 5 μl (25 μg) was taken and mixed with 10 μl of sample buffer for electrophoresis.

Bovine Serum Albumin (BSA) fraction-V

BSA (1 mg) was weighed and dissolved in 1 ml of double distilled water (stock). From this 3 μl (3 μg) was taken and mixed with 10 μl of sample buffer for electrophoresis.

Haemoglobin from Ox RBC

Ox RBC was collected from the Corporation Slaughter House, Perambur, Chennai. It was collected in Alsever’s solution. The RBC was washed thrice with 0.9% physiological saline by centrifugation at 500×g for 10 min. From the suspension, 10 μl of RBC was taken and diluted to 2 fold with 25 mM NaCl. This solution was incubated for 30 min at Room Temperature (RT), centrifuged at 10,000 rpm for 15 min. From this 2 μl (28μg) of supernatant was directly loaded on the gel.

Haemolymph

A healthy fifth instar silkworm (Bombyx mori) larva was wiped on the ventral portion with ethanol. With the help of sterile scissors, the foreleg was cut and the oozed out haemolymph was collected in vial containing Phenylthiourea (PTU) to prevent melanisation. It was centrifuged at 10,000 rpm for 10 min. The supernatant (1 μl equivalent to 18 μg of protein) was directly loaded on the gel.

Analysis of protein samples in Native-PAGE

The protein samples were analyzed using native PAGE [17]. The gel plates were assembled according to the manufacturer’s instruction and the volume of the gel mould was determined (170 x 150 x 1.5 mm). In a beaker, the resolving gel mixture for 8% (2.70 ml of 30% acrylamide stock + 2.50 ml of 1.5 M Tris HCl (pH 7.8, 8.3, 8.8 and 9.3), + 4.60 ml of double distilled water + 100 μl of 10% ammonium per sulphate + 6 μl of TEMED) was prepared. The components were mixed thoroughly and without delay the resolving gel mixture was poured into the glass mould and overlaid with distilled water carefully. After polymerization (approximately 20 minutes), the overlay was removed. 5% stacking gel was prepared by mixing 0.67 ml of 30% acrylamide stock, 0.50 ml of 0.5 M Tris HCl, pH 6.8, 2.70 ml of distilled water, 40 μl of 10% ammonium per sulphate and 4 μl of TEMED. These components were mixed and poured over the resolving gel. Immediately a comb was inserted and the gel was allowed to polymerize (approximately 15 min). Once it got polymerized, comb was removed and lanes were washed with distilled water. The gel mould was placed in an electrophoresis apparatus and completely filled with electrophoresis (tank) buffer (pH 8.3, 8.8 and 9.3).

Using a gel loading tips, lysozyme (25 μg) was mixed with 10 μl of sample buffer and loaded in the I lane. Ox haemoglobin (20 μg) was mixed with 10 μl of sample buffer and loaded in the II lane. BSA (3 μg) was mixed with 10 μl of sample buffer and loaded in the III lane. Haemolymph (18 μg) was mixed with 10 μl of sample buffer and loaded in the IV lane.

Electrophoresis at different combinations of pH in electrophoresis (tank) and resolving gel buffers.

In one set of experiment, the pH of the resolving gel was kept constant at 7.8 and the pH of the electrophoresis buffer was changed to 8.3, 8.8 and 9.3. In the second experiment, the pH of the resolving gel was kept at 8.3 and the pH of the electrophoresis buffer was changed to 8.3, 8.8 and 9.3. In the third experiment, the pH of the resolving gel was kept at 8.8 and the pH of the electrophoresis buffer was changed to 8.3, 8.8 and 9.3. In the fourth set, the pH of the resolving gel was kept at 9.3 and the pH of the electrophoresis buffer was changed to 8.3, 8.8 and 9.3.

The apparatus was plugged to the power supply unit taking care of anode and cathode wire plugged properly. Initially the voltage was set at 60 for stacking gel and later 120V for resolving gel. The power supply was turned off, once the bromophenol blue reached the bottom of the resolving gel. The gel mould was removed apart using a spatula. The orientation of the gel was marked. The gel was stained with staining solution. Each box was labelled for identification. After 4 hours, each gel was washed with distilled water and destaining solution was poured. Destaining solution was changed until the background was cleared. It was stored in 7% acetic acid. The bands were visualized using an illuminator and then photographed.

Results

Silkworm haemolymph sample, hemoglobin from ox RBC and two commercially purchased proteins such as lysozyme and BSA were used in the study to show the influence of pH in the buffer systems used in native-PAGE. The results obtained were presented in the order of constant pH of resolving gel buffer with differing pH of tank buffers. At a resolving gel buffer of pH 8.8, three different pH of electrophoresis or tank buffers (pH: 8.3, 8.8 and 9.3) were used to separate various samples in the acrylamide gel. The native gel system comprised of resolving gel pH 8.8 with a combination of tank buffer pH 8.3 separated lysozyme, haemoglobin, BSA and the silkworm haemolypmph proteins better than other pH variants of tank buffers (i.e., pH 8.8 & 9.3) (Figure 1A). The increasing pH of tank buffers made proteins to diffuse over the gel and separate poorly in native- PAGE (Figures 1B and 1C).

    

    

    Figure 1: Protein profile of various samples in Native-PAGE (RGB –
Resolving Gel Buffer; TB – Tank Buffer). (A) RGB - pH 8.8, TB – pH 8.3 (B)
RGB - pH 8.8, TB – pH 8.8 (C) RGB - pH 8.8, TB – pH 9.3 (D) RGB - pH 7.8,
TB – pH 8.3 (E) RGB - pH 7.8, TB – pH 8.8 (F) RGB - pH 7.8, TB – pH 9.3 (G)
RGB - pH 8.3, TB – pH 8.3 (H) RGB - pH 8.3, TB – pH 8.8 (I) RGB - pH 8.3,
TB – pH 9.3 (J) RGB - pH 9.3, TB – pH 8.3 (K) RGB - pH 9.3, TB – pH 8.8 (L)
RGB - pH 9.3, TB – pH 9.3. I. Lysozyme (14.6 kDa, pI 11); II. Haemoglobin
(64 kDa, pI 7.3); III. BSA (66.3 kDa, pI 4.9); IV. Silkworm haemolymph.