• Home
  • Search Results
  • Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis.

Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis.

Proteome science (2011-10-13)
Carl Pc Chen, Chih-Chin Hsu, Wen-Lin Yeh, Hsiu-Chu Lin, Sen-Yung Hsieh, Shih-Cherng Lin, Tai-Tzung Chen, Max Jl Chen, Simon Ft Tang
ABSTRACT

Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone. The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, cold ethanol fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, suitable for RIA, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, lyophilized powder, essentially fatty acid free, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, crystallized, ≥98.0% (GE)
Sigma-Aldrich
Bovine Serum Albumin, pH <5.0, powder
Sigma-Aldrich
Bovine Serum Albumin, powder, BioXtra
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, suitable for hybridization, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, low endotoxin, lyophilized powder, Cohn Analog, BioReagent, suitable for cell culture, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free and essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, Cohn Fraction V, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 5.2, ≥96%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially IgG-free, low endotoxin, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, BioReagent, suitable for cell culture
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, low endotoxin, ≥98% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, 1× crystallized, ≥97% (agarose gel electrophoresis)