5hrs with horseradish peroxidase conjugated secondary antibody. The protein bands were found by an enhanced chemiluminesence set. Nude natural product library mice xenografts Androgen dependent LNCaP and independent LNCaP AI prostate cancer cells, combined with Matrigel in a rate of 1:1 were inoculated to the flanks of 4 5 week old male Nu/Nu Balb/c athymic nude mice by subcutaneous injection. every 3 days the cyst growth and size was checked. Animals were randomly split into 2 teams, 10 mice each, in accordance with tumor size, when the prostate tumor grew to a diameter of 4 8 mm. One group of animals was treated with drug vehicle just as control, and yet another group was treated with Natura alpha at amount of 100mg/kg by gavage, once every day, 5 days per week before size of tumors in control group reached approximately 15 mm. The tumor growth was monitored day-to-day and tumor size recorded every three days. The tumor volume was determined as l x n x h x 0. 52. Proteomic Pathway Array Analysis Total cellular proteins were extracted from tumors using a lysis buffer containing 20mmol/L sodium Pyrophosphate, 20mmol/L Tris HCl, 40mmol/L T physical form and external structure glycerophosphate, 30mmol/L Sodium Fluoride, 2mmol/L EGTA, 10mmol/L NaCl, and 0. Five hundred NP 40. The lysate was sonicated three times for 15 seconds everytime, and then centrifuged. The tubes were kept on ice throughout the process. The protein concentration was determined using the BCA Protein Assay kit. Isolated proteins were separated by SDS PAGE. Three hundred ug of protein extracts were loaded in a well over the whole thickness of gel for SDS PAGE, followed by electro transferring to a nitrocellulose membrane. The membrane was then blocked for 1 hr with five minutes milk or a few months BSA, and held on to a Mini PROTEAN II Multiscreen device that isolates 20 channels over the membrane. 2 or 3 antibodies were put into each station and incubated over night at 4oC. Various sets of antibodies were employed for each membrane after stripping supplier CX-4945 the last group of antibodies. Antibodies were acquired both from Cell Signaling Technology, Inc. or from Santa Cruz Biotechnology, Inc.. The pathway range analysis was run in duplicate for each test in each set of antibodies and protein levels were normalized applying GAPDH and beta actin as criteria. Chemiluminescence signals were captured using the ChemiDoc XRS System. Variations in protein levels were determined by densitometric scanning and normalized to internal standards. IRB and fda permitted single patient clinical trial A 86-year old patient with higher level androgen independent metastatic prostate cancer was consented for the Natura alpha trial treatment for his condition with approval from the IRB and FDA. Natura leader was administered orally with increasing doses from 40mg, 80mg, 160 to 200 mg daily every two weeks and 200 mg afterwards for three months.