INSTA-Blot™ Breast Tissue OncoPair

Introduction

The development of biomarkers has enormous potential for revolutionizing the diagnosis and treatment of disease. Western blot analysis is an integral technique for biomarker profiling. While key for biomarker discovery, the existing body of western blot data has primarily been defined from studies of tumor, immortal, and primary cells growing in vitro. Collectively, results obtained over decades have been integral to the dogma that up- and down-regulation of proteins can be leveraged as biomarkers of normal development, homeostasis, and disease. However, data from human tissue-derived products is key for generating biomarker validation data and progressing the most promising biomarker targets towards the clinic. The OncoPair INSTA-Blot^TM product line addresses the increasing demand for the inclusion of human tissue-derived products in biomarker profiling.

The OncoPair INSTA-Blot^TM is a ready-to-use PVDF western blot membrane containing denatured protein lysates manufactured from Imgenex’s extensive Human Clinical Tissue Lysate collection. Each blot contains 14 lanes of alternating tumor (T) and matched normal adjacent (NA) tissue lysates from 7 patient donors. Incorporating both T and NA on the blot enables protein expression analysis of the tumor and microenvironment. The tumor microenvironment is increasingly being recognized as a major factor in influencing malignant progression and metastatic potential.

Cancer is a heterogeneous disease and the presence of multiple donors on a single blot enables rapid screening of protein expression variability between T/NA from different individuals and at different disease stages. Clinical diagnosis and histopathology reports from board-certified pathologists are available for every patient sample. OncoPair INSTA-Blots^TM are available for a wide range of tumor types and stages. The product line is manufactured from donor patient tissue obtained from established hospital-based collection sites around the world. Tissues are collected under strict bioethical standards using IRB and HIPAA approved protocols. These protocols ensure patient confidentiality, safety, and informed consent. Tissues are flash frozen at collection sites within minutes of removal, maintained in liquid nitrogen and processed into lysates using protocols optimized for extracting proteins from tissues. Lysates are denatured for OncoPair INSTA-Blot^TM production; native lysates are available for follow up proteomic studies. OncoPair INSTA-Blots^TM and lysates are also useful in conjunction with the Cell Line and Normal Tissue INSTA-Blots^TM and lysates for comprehensive antibody validation and to profile antibody reactivity and protein expression patterns across species and between tissues and cells lines. The OncoPair INSTA-Blots^TM are proteomic discovery tools and are useful for screening the expression of various proteins in tumor and normal adjacent tissues from various donors. Researchers may want to follow up on their results by further analysis with the corresponding individual lysates when available.

Preparation

Tissue specimens were homogenized in modified RIPA buffer to obtain the soluble proteins and centrifuged to clarify. The concentration of protein in each lysate was determined by a standard protein assay and standardized to 1 mg/ml. Sample buffer was added to the soluble fraction and approximately 14 ug/lane of protein was run and then transferred to PVDF membrane.

· Soluble fraction extraction buffer (extract 1): PBS at pH 7.4, 1 µg/ml Aprotinin, 1 mM NaF -Modified RIPA buffer: 1 mM EDTA, 1 µg/ml Pepstatin-A, 0.1% SDS , 0.25% Na deoxycholate, 1 µg/ml Leupeptin, 1 mM PMSF, 1 mM Na3VO4

· 5X Sample buffer: 50 ml glycerol, 15 g SDS, 3.819 g Tris, pH 6.8, 25 ml 2-ME, 100 mg bromophenol blue, final volume of 100 ml with DI H2O.

Protocols

Note: The INSTA-Blot^TM PVDF membrane has been dried and must be rehydrated (Step one) prior to use.

Before wetting, it is suggested that the top of the lanes be marked with an ink pen that will not wash off in methanol.
1. Wet the blots with 100% methanol then thoroughly wash with TBST (25 mM Tris-Cl, pH 8.0; 125 mM NaCl; 0.1% Tween 20) twice to remove residual methanol.
2. Incubate the blot for 1 hr with 5% Carnation nonfat dry milk in TBST to block non-specific antibody binding.
3. Incubate the blots with primary antibody in 1% milk/TBST for 1-2 h at RT or overnight at 4°C.
4. After incubation with the primary antibody, wash the blots five times in TBST then incubate with a secondary antibody conjugated to horseradish peroxidase (IMGENEXs HRP-conjugated secondary antibodies) for 1-2 h at RT.
5. After five washes with TBST, develop the blots for 5 min using the PicoTect^TM Western Blot Chemiluminescent Substrate (IMGENEX, Cat. No. 10087K).
6. Expose the blots to photographic film for an appropriate time period. We normally use Hyper-film^TM-ECL films (Amersham Life Science Inc.) and expose for various periods ranging from 10 s to 20 min to visualize the chemiluminescence signal corresponding to the specific antibody-antigen reaction.

Reference
1. Expression, Tissue Distribution, and Cellular Localization of the Antiapoptotic TIP-B1 Protein. Erica S. Berleth, Patricia A. Masso-Welch, Latif A. Kazim, Margot M. Ip, Enrico Mihich, and M. Jane Ehrke. J. Leukoc. Biol., 69: 995-1005 (2001).