Study of extraction procedures for protein analysis in plankton samples by OFFGEL electrophoresis hyphenated with Lab-on-a-chip technology.
Extraction procedures for protein analysis from plankton samples were studied. OFFGEL electrophoresis combined with Lab-on-a-chip technology has been applied for protein analysis in plankton samples. BCR-414 (plankton) certified reference material from the European Commission was used to evaluate the protein extraction procedures. Three protein extraction procedures were studied: (1) by using Tris-HCl buffer containing a protease inhibitor cocktail, (2) urea/triton X-100 buffer extraction, and (3) using the phenol/sodium dodecyl sulphate method after different washing steps with 10% trichloroacetic acid/acetone solution and methanol. The pellet of proteins obtained was dried and then dissolved in the OFFGEL buffer. Proteins were separated according to their isoelectric points by OFFGEL electrophoresis. This separation was performed using 24 cm, pH 3-10 IPG Dry Strips. The proteins present in each liquid fraction (24 fractions) were separated according to their molecular weight using a microfluidic Lab-on-a-chip electrophoresis with the Protein 80 LabChip kit. This kit allows for the separation of proteins with a molecular weight ranging from 5 to 80 kDa. Taking into account the intensity and the number of the protein bands obtained, the protein extraction procedure using the phenol/sodium dodecyl sulphate after different wash steps with 10% trichloroacetic acid/acetone solution was selected. The developed method was applied for protein determination in a fresh marine plankton sample. The proteins found in this sample have a molecular weight ranging from 6.4 to 57.3 kDa, and the proteins with highest molecular weight were in the OFFGEL fractions with an isoelectric point ranging from 4.40 to 8.60. The concentration of proteins were calculated using external calibration with Bovine Serum Albumin, and the protein concentrations varied from 50.0 to 925.9 ng µL(-1).
Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Natalia
N
García-Otero
Ma Carmen
MC
Barciela-Alonso
Antonio
A
Moreda-Piñeiro
Pilar
P
Bermejo-Barrera
D000096:Acetone; D002138:Calibration; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005060:Europe; D007526:Isoelectric Point; D046210:Microfluidic Analytical Techniques; D008970:Molecular Weight; D019800:Phenol; D010933:Plankton / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D012015:Reference Standards; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid
D011480:Protease Inhibitors; D011506:Proteins; D000096:Acetone; D019800:Phenol; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid
Lab-on-a-chip technology; OFFGEL electrophoresis; Plankton samples; Protein extraction