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
If this protocol has papers or books associated with it, list those references here. Below is an example for formatting purposes. See the OpenWetWare:Biblio page for more information.
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
- ISBN:0879697164
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