INSTA-Blot™ Breast Tissue OncoPair Set

Introduction
OncoPair INSTA-Blots^TM are ready-to-use PVDF western blot membrane containing denatured protein lysates manufactured from IMGENEX's extensive Human Clinical Tissue Lysate collection.  The IMB-130set OncoPair Breast Set contains 5 ready-to-use Breast OncoPair INSTA-Blots^TM. The donor patient tissues in this Set collectively represent a range of breast tumor types, stages, and patient ages. The blots are themed by breast cancer type or stage, each blot contains 14 lanes of alternating tumor (T) and matched normal adjacent (NA) tissue lysates from 7 patient donors. There are 34 different breast cancer patient donors represented in the 5 Blot Set, with one patient donor repeated. Refer to each individual INSTA-Blot^TM catalog number link for cancer type, stage, and age of patient donors.
Additional diagnostic details for each patient is available on the IMGENEX Website (www.imgenex.com) by the lysate catalog numbers provided on each OncoPair INSTA-Blot^TM. The OncoPair INSTA-Blot^TM product line addresses the increasing demand for the inclusion of human tissue-derived products in biomarker profiling, and the 5 Blot Set provides expanded screening opportunities compared to using one blot alone. Blots and lysates are also available separately for follow up studies. Following initial screening, researchers are encouraged to obtain sufficient quantities of the blots and lysates they need to carry out their studies as all human-derived material may be available in limited supply.

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.

· 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.