Detection of Flag-tagged RASSLs by ELISA in Tissue Culture Cells

Overview
List of reagents is not yet complete

Procedure
Day 1: Seed Cells

1. Seed HEK 293 at 20,000 cells/well (~1ml) on a 24-well plate.

Day 2: Transient Transfection

1. Prepare DNA-Superfectant complex (amount per 24-well; prepare enough for triplicate)

DNA (1ug/well) 1ug

DMEM (no FBS) 60 ul

Superfectant (1ugDNA/5ul Supf) 5 ul

2. Vortex, incubate at RT for 10 min

3. Wash cells once with 0.5 ml DMEM (no FBS)

4. Add 300ul DMEM/FBS to cells

5. Add DNA-Superfectant complex to cells.

6. Incubate for 2 hrs at 37°C.

7. Aspirate media. Add 0.5ml fresh media to each well.

Day 4: Agonist Stimulation

Day 4 or 5: Plate Assay

This protocol is for detecting proteins on surfaces of any adherent cell with antibody by ELISA-like method.

1. Discard media into sink. Put the plate upside-down onto a blue pad,Press.

2. Add 0.25 ml / well of cold 2-4% PFA in PBS (10 mM Na-phospahte, pH7.0; 150 mM NaCl), for Immunex flag. (Always gently! Do not inject directly to cell surface).

3. Fix for 10 min at 4°C.

4. Wash twice with 0.5 ml of PBS (regular PBS - pH 7.4)

5. Add 0.25 ml / well of 1° antibody (anti-Flag antibody; final 1ug/ml)in The Media.

6. Incubate for 1 hr at room temperature. (begin dissolving ABTS now- see step 9)

7. Gently rinse once with 0.5 ml / well 1x PBS + 1 mM CaCl2.

8. Add 0.25 ml / well 2° antibody, (Horse Radish Peroxidase - GoatAnti-Mouse) 1:1000 in the Media. Incubate for 30 min at room temperature.

9. Prepare developing solution:

1mg / ml ABTS in

1x Citrate/phosphate buffer, pH 4.0 (0.1 M NaCitrate, 0.1 M Na2HPO4 final),

plus H2O2 1ul / ml - add H2O2 last, just before using solution

(Use fitered 2x Citrate/phosphate buffer stock; prep from NaCitrate powder,85% O-H3PO4, pH with conc Hcl)

10. Rinse three times, 5 min each with 0.5 ml / well 1x PBS + 1 mM CaCl2. (Swirl the plate gently when rinsing).

11. Add 0.25 ml / well of developing solution to each well.

12. Wait 10 min - 1 hr at room temperature for the development.

13. Take 200 ul from each well and transfer to well of a 96 well plate.

14. Read the OD on plate reader at 410nm or as close as possible.

The Media - DMEM + 10% FBS + 1 mM CaCl2

Antibody Wash Solution - 1x PBS - pH 7.4 + 1 mM CaCl2

Anti-Flag Antibody (M1) - Eastman Kodak Co. # IB13007

HRP-GAM - BioRAD Cat # 170-6516

ABTS - SIGMA Cat # A-1888

H2O2 - SIGMA Cat # H-1009

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