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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Purification of Bacterially Produced His-tagged Proteins on a Nickel Column

Purification of Bacterially Produced His-tagged Proteins on a Nickel Column Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Bruce Conklin, The Gladstone Institute at the University of California, San Francisco. URL: Lab Web Site
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Cells are transfected with a FLAG-tagged cell surface receptor gene. Following agonist stimulation, anti-FLAG antibodies are used to detect the receptors in a technique that is similar to Enzyme-linked immunosorbent assays (ELISAs).
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Transfect Cells 1. Seed appropriate number of 1 ml aliquots of HEK293 cells (Human Embryonic Kidney cell line) at 20,000 cells/well into the wells of a 24-well tissue culture plate and incubate the plate overnight. 2. Prepare DNA-SuperFect Transfection Reagent complex for transient transfection by mixing the following components in a microcentrifuge tube (mixture amount is per well of cells, see Hint #2): 1 μg of DNA 60 μl of DMEM minus FBS 5 μl of SuperFect Transfection Reagent Scale up the amounts accordingly for total number of transfections. 3. Vortex to mix and incubate at room temperature for 10 min. 4. While mixture is incubating, wash the cells with DMEM minus FBS by aspirating the medium from each well, adding 0.5 ml of DMEM minus FBS to each well, and aspirating the medium from the wells again. 5. Add 300 μl of DMEM plus FBS to each well of cells. 6. Add the DNA-SuperFect complex to each well of cells. 7. Incubate cells with DNA-SuperFect complex for 2 hours at 37°C in the incubator. 8. Aspirate the medium from each well and add 0.5 ml of DMEM plus FBS to each well of cells. 9. Incubate for 2 to 3 days in the incubator. B. Detection of Receptor Expression by Plate Assay 1. Stimulate expression of the tagged receptor by exposing cells to the appropriate agonist. 2. Remove media from cells and place the plate upside-down onto absorbent material (i.e. absorbent bench padding). 3. After residual media is absorbed away from the plate, gently add 0.25 ml of ice-cold Paraformaldehyde Solution (see Hints #3 and #4). 4. Incubate for 10 min at 4°C. 5. Aspirate the supernatant from the wells and add 0.5 ml of PBS. Aspirate the PBS from the wells. 6. Add 0.5 ml of PBS to each well again, and aspirate the PBS again from the wells. 7. Add 0.25 ml of Primary Antibody Solution to each well. 8. Incubate the cells with antibody for 1 hour at room temperature. 9. While cells are incubating, prepare the ABTS Solution. 10. After incubation, remove the solution from the wells and wash the cells with 0.5 ml of PBS plus 1 mM CaCl2 to each well (see Hint #5). 11. Add 0.25 ml of Secondary Antibody Solution to each well. 12. Incubate cells in Secondary Antibody Solution for 30 min at room temperature. 13. Remove the solution from the wells and wash the cells with 0.5 ml of PBS plus 1 mM CaCl2 to each well. 14. Repeat Step #13 two more times. 15. Add 1 μl of Hydrogen Peroxide per ml of the ABTS Solution. 16. Add 0.25 ml of activated ABTS Solution to each well. 17. Wait the appropriate amount of time (approximately 10 min to 1 hour) for the development of the reaction product from the ABTS Solution. A blue green product will appear. 18. Take 200 μl from each well and transfer to individual wells of a 96-well plate. 19. Read the Absorbance at 410 nm wavelength in a spectrophotometer plate reader.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Secondary Antibody Solution    1 mM CaCl2 1:1000 diluted Goat Anti-Mouse Antibody conjugated to Horse Radish Peroxidase DMEM plus FBS ABTS Solution    pH 4.0 0.1 M Na2HPO4 1 mg/ml ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic Acid)) 0.1 M Sodium Citrate Primary Antibody Solution    in DMEM plus FBS 1 mM CaCl2 1 μg/ml of Anti-FLAG Antibody 1 mM CaCl2 PBS    4.3 mM Sodium Phosphate, Dibasic (Na2HPO4) pH 7.2 2.7 mM KCl 1.8 mM Potassium Phosphate, Monobasic (KH2PO4) 137 mM NaCl Paraformaldehyde Solution    10 mM Sodium Phosphate, pH 7.0 2% to 4% (w/v) Paraformaldehyde (CAUTION! see Hint #1) 150 mM NaCl DMEM plus FBS    10% (v/v) Fetal bovine serum (FBS) DMEM DMEM minus FBS    Dulbecco's Modified Eagle's Medium (DMEM) Plasmid DNA    DNA must contain FLAG-tagged gene of interest
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
SuperFect Transfection Reagent Potassium Phosphate, Monobasic Goat Anti-Mouse Antibody conjugated to Horse Radish Peroxidase Paraformaldehyde Sodium Phosphate, Dibasic Fetal Bovine Serum Dulbecco's Modified Eagle's Medium Potassium Chloride Anti-FLAG Antibody Sodium Chloride 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic Acid) Hydrogen Peroxide Calcium Chloride Sodium Phosphate Sodium Citrate
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Prepare enough of the mixture for triplicates (i.e. perform three separate transfections for every sample). 3. The optimal concentration of Paraformaldehyde to use (between 2 to 4%) should be determined empirically for the particular tagged receptor protein. 4. Add fixative down the sides of the wells, do not squirt directly on cells. 5. To wash the excess antibody from the wells, after adding the PBS solution, swirl the solution in the wells gently for a few seconds before aspiration.