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
Notes
No further notes are available at this time.
References
Relevant papers and books
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