Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis.
Sample pre-treatment is a critical step for an efficient and reliable analysis and it is highly dependent on the complexity of the matrix. This work shows an example of application of an immunoprecipitation approach using a new magnetic beads-based format, which allows a selective/specific extraction of potential biomarkers from metastatic prostate cancer. Results obtained on the development of this method, and its application for the extraction and pre-concentration of certain biomarkers present in metastatic cell lines of prostate cancer, are presented and discussed. It is concluded that the efficiency of the immunoprecipitation step is clearly compromised by the crosslinking conditions and it is highly dependent on the specificity of selected antibodies. The epoxy magnetic beads used in this work allowed an effective crosslinking of the antibodies contributing to an increased efficiency of the immunoprecipitation step. The optimized conditions for the application of these epoxy magnetic beads for the immunoprecipitation of anti-TUBA3C in metastatic prostate cancer cell line (PC3) are discussed here, as an example of application of the immnuprecipitation approach developed, which resulted in a very efficient tool for a specific extraction and pre-concentration of the targeted protein and, therefore, contributing to the efficiency of further analysis.
Vila
A Molares
AM
Department of Analytical Chemistry, Chemistry Faculty, University of Vigo, Vigo, Spain.
de Arrilucea
P Rupérez Pérez
PR
Peláez
E Caso
EC
Gago-Martínez
A
A
D000912:Antibodies, Neoplasm / Q000032:analysis; D014408:Biomarkers, Tumor / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D047468:Immunoprecipitation; D008280:Magnetics; D008297:Male; D011471:Prostatic Neoplasms / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000032:analysis / Q000276:immunology
D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor; C408046:TUBA8 protein, human; D014404:Tubulin