Refolding Protocol for Ep23, 5'-DY647

This is intended as a guide only; for full experimental details please read the reference provided.

Binding Data

Ep23, 5’-DY647 (Cat.No. 6102) as a Kd of 39.42 nM against EpCAM as measured by flow cytometry (see figure below for binding data in HT29 cells).

Binding Data

Reconstitution and Refolding Protocol

Reconstitute the aptamer at 100 µM in sterile deionized H2O and ensure the aptamer has fully dissolved. We recommend that stock solutions, once prepared, are stored aliquoted in tightly sealed vials at -20°C or below and used within 1 month. Wherever possible solutions should be made up and used on the same day. Repeated freeze thaw cycles should be avoided.

Prior to use, dilute the aptamer at an assay dependent concentration in PBS buffer containing 5 mM MgCl2. Heat the solution at 85˚C for 5 min, incubate for 10 min at room temperature and finally allow to refold for 15 min at 37˚C.

Unfixed Cell Imaging Protocol

(This protocol is also applicable to flow cytometry)

Twenty-four hours prior to labeling, seed cells at a density of 75,000 cells per cm2 in an 8-chamber slide (Lab-Tek II, Nunc). Remove media and incubate cells in blocking buffer (PBS buffer containing 5 mM MgCl2, 0.1 mg/mL tRNA (R5636, Sigma), and 5% FBS) at 37 ˚C for 15 min. Wash twice in binding buffer (DPBS with 5 mM MgCl2, 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA) prior to incubation with 200 nM (1/500 dilution) refolded aptamer (typically 100 µL) for 30 min at 37 ˚C. Additional step for unfixed cell imaging only: add Hoechst 33342 (3 µg/mL) to the cells during the final 15 min of incubation. Remove the aptamer solution and wash three times for 5 min each in binding buffer prior to visualization.

Reagent Tocris Cat. No.
PBS 5564
Hoechst 33343 5117

Fixed Tissue Imaging Protocol

Deparaffinize paraffin embedded sections with Histoclear and rehydrate through graded ethanols. Perform heat induced antigen retrieval in a microwave oven using Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween® 20, pH 9.0) for 20 min and allow slides to cool prior to blocking with 0.1 mg/mL tRNA (R5636, Sigma), and 1 mg/mL bovine serum albumin or 10% goat serum in phosphate buffered saline (PBS) for 1 h. Following the blocking step, wash slides in PBS containing 0.1% Tween® 20 twice for 2 min prior to incubation with aptamer. Refold Ep23, 5’-DY647 using the refolding procedure above, and apply to tissue at a concentration of 100 nM in PBS containing 5 mM MgCl2, 0.1 mg/mL tRNA, 1 mg/mL BSA, 10% dextran sulfate and 500 mg/mL heparin for 15 min at 37˚C. Wash slides in PBS containing 0.1% Tween® 20 three times for 5 min each prior to incubation with Hoechst 33342 (3 µg/mL) for 10 min. Mount slides using VECTASHIELDH (Vector Laboratories, Burlingame, CA) and apply coverslips. N.B: The aptamer must be refolded according to the above procedure prior to the addition of 0.1 mg/mL tRNA, 1 mg/mL BSA, 10% dextran sulfate and 500 mg/mL heparin.

This protocol has successfully been used for IHC EpCAM staining in the following xenograft tumors: T47D, MCF7, MDA-MB-231 (breast cancer) and HT-29 (colon cancer).

Reagent Tocris Cat. No.
EDTA 2811
Heparin Sodium Salt 2812
TRIS hydrochloride 3164
Hoechst 33343 5117
Bovine Serum Albumin 5217
PBS 5564

Tween is a registered trademark of ICI Americas.

Unfixed Tumorspheres Imaging Protocol

Wash spheres three times in PBS containing 5 mM MgCl2 and block for 20 min using blocking buffer (PBS buffer containing 5 mM MgCl2, 0.1 mg/mL tRNA and 5% FBS). Incubate spheres with 100 nM refolded Ep23, 5’-DY647 for a minimum of 30 min. Wash spheres three times with PBS prior to visualization.

This protocol has been produced in collaboration with Dr Sarah Shigdar, Deakin University, Australia.

Reference

Shigdar et al. (2011) RNA aptamers against a cancer stem cell marker epithelial cell adhesion molecule. Cancer Sci. 102 991. PMID: 21281402