For multiple proteins containing fractions, we planned to perform 2D-gel electrophoresis as part of the second phase of study. Using the relatively simple and cost-effective technique of 1D SDS PAGE http://www.selleckchem.com/products/ganetespib-sta-9090.html in combination with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), we have identified apolipoprotein A1 (apoA-1), the Ig kappa (��) chain C region, and transthyretin (TTR), along with some other acute phase proteins (eg, haptoglobin (Hp), serum amyloid A (SAA), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), serum albumin) and some components of a complement system (i.e, C8, C3 and C4) as potential biomarkers for breast carcinoma.

Materials and Methods Sample collection After approval from the Institutional Ethical Committee, blood samples (n = 61) were collected from patients presenting at Bahawal Victoria (BV) Hospital, Bahawalpur, Pakistan, following standard procedures and written consent taken from each patient on a prescribed form. Complete demographic, disease and medication history of each patient was recorded in the questionnaire. Collected sera were first screened for HBV/HCV/HIV infection. No sample was found to be HIV positive. However, few samples were positive for HBV (n = 1) and HCV (n = 7). A fraction of BC patients (n = 8) had undergone chemotherapeutic treatment. The patients found positive for HBV/HCV infection and those who had been treated chemotherapeutically were excluded from the main cohort of samples. Excluded samples were treated as a separate group and analyzed to get an idea about combinatorial effect of BC and either HBV/HCV infection or chemotherapy.

Female patients with BC (n = 48) and non-malignant pre-cancerous benign breast lesions (n = 13) were included in the study. The mean age of the patients was 45.54 �� 13.15 years. Sample processing and protein gel electrophoresis Sera obtained using standard methodology were stored at ?70 ��C in small aliquots until further analysis took place. Proteins were quantified using the Bradford method.31 Following the optimized gel conditions, 60 ��g of serum proteins were resolved on 10% SDS PAGE.32 Gels were run for 2 hours (h) at 125 volts and stained using 0.25% solution of Coomassie brilliant blue (G-250) for 2 h. Sera of normal healthy female subjects were used as a normal control comparison. Differentially expressed protein bands, compared with the sera of normal individuals, were cut from the gel and identified using LC-MS/MS.33 GSK-3 Scores of peptide match and protein sequence coverage% (Table 1) provided an account of the degree of confidence associated with the identification of proteins by mass spectrometry. A higher peptide match and protein sequence coverage score is the indicator of higher confidence with which the protein is identified.