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Abstract

Objective: It seems that the type of buffer for extraction of proteins from each sample is important. However, the most widely used buffer in these researches is rehydration buffer. In this study, we compared the 2 dimensional electrophoresis (2-DE) maps from different buffers.

Materials and methods: 2-DE map of rat testicular tissue obtained by three distinct buffer included phosphate buffer saline (PBS (A), Tris-HCl (B), or lysis buffer (C) were evaluated and compared.

Results: 2-DE map of rat testicular tissue obtained by three distinct buffer included phosphate buffer saline (PBS (A), Tris-HCl (B), or lysis buffer (C) were evaluated and compared.

Conclusion: Our results demonstrated that using buffer 0.05 M Tris-HCl, 10% sucrose, pH 6.80 was provided good comparable results with rehydration buffer. However, buffer A was not appropriate for 2-DE. Due to the expensiveness of CHAPS and ampholite, which were used in the rehydration buffer, the replacing of it with buffer B that produced good 2-DE is recommended.

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Introduction

Appropriate sample preparation is critical for obtaining high quality two-dimensional electrophoresis (2-DE) separations and reliable results in a proteomic analysis [1,2].The first step of sample preparation is the effective disruption of the cell membrane to release the entire array of proteins followed by centrifugation at relatively high speed. Before that, sample solution must be prepared by using appropriate buffer. Various buffers with different compositions, ionic strength, molarity and pH value were used, but deciding about the selection of certain buffer is still difficult. The 2-DE sample rehydration buffer (RHB) is widely used to denature and solubilize protein samples. On the other hand, phosphate saline (PBS) and Trishydrochloride (Tris-HCl) buffers are used for sample preparation for 2-DE and other analytical methods. In this study, we compare 2-DE gel patterns obtained by rat testes tissue preparation using the three conventional distinct buffers, Tris-HCl, PBS and RHB.

Urea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), iodoacetamide, Tris, acrylamide, methylene bisacrylamide, sodium dodecyl sulphate (SDS), ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), glycerol, DTT, 2 mercapto ethanol (2 ME), Bromophenol blue (BPB) was purchased from Sigma. IPG strips (7 cm length) werepurchased from Bio-Rad, Hercules, CA,USA. Sodium monophosphate, hydrochloric acid (HCl) and sodium hydroxide were purchased from Merk.

One fourteen-weeks old male Sprague Dawley rat was selected. The animal was sacrificed by cervical dislocation and testes was immediately removed. Left testes frozen and stored in -70 °C until sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDSPAGE) and 2DE gel analysis.

Left testes from one fourteen-weeks old male Sprague Dawley rat was used. Three hundred mg of testis tissue was placed in a crucible on ice along with 6 Vol of a solution A (0.1 M phosphate buffer saline, pH 7.4), B (0.05 M Tris-HCl, 10% sucrose, pH 6.80), or C (8 M urea, 2% (v/v) CHAPS, 2% (v/v) immobilized pH gradient (IPG) buffer (pH 3-10 NL), and 2% (w/v) DTT). The tissue was thoroughly minced with surgical scissors and then grounded manually under liquid nitrogen. The homogenates were centrifuged at 14000 rpm for 15 min at 4 °C to remove tissue and cellular debris and supernatents were used as the source of protein. Protein concentration was determined according to the method proposed by Bradford, using BSA as a standard protein [3]. 2-DE was performed according to our previous reported procedure [4,5] using IPG 3-10 NL, 7 cm in 1st dimension and 12% acrylamide gels as 2nd dimension at 4 °C by using up to 1 mg protein per each strip. Immediately after electrophoresis, commassie brilliant blue staining was performed to determine differences in the protein pattern on 2-DE maps. All experiments were performed as triplicate and respected results are reported as mean and standard deviation (SD). SPSS version 20.0 was used for statistical analysis. Comparison of spot numbers and also the percentage of spot’s volume and intensity between three extraction buffers were performed using Kruskal-Wallis test.

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Results

The protein content of samples provided by different buffers show no significant difference. The number of protein spots in the 2-DE map by using of buffers B and C is approximately 2 times more than buffer A (p< 0.05). Also, the most of these spots were appearing in the slightly alkaline region (pI 7.5-9.0) from all molecular weight ranges in buffers B and C. By using buffer A, the most spots were in the neutral pI (5.5-7.5) and more appeared in the low range of molecular weight (Table 1).

Although the percentage of volume and intensity of the detected spots in the Tris-HCl and rehydration buffers were higher than PBS buffer (Table 1), but, the spots from all three buffers were sharp (clear edges) with good intensity and volume (Figure 1) and there were no significant differences neither in %volume, nor in %intensity between three groups.