ATP Filter Binding

 

Adapted from: The European Working Group on CFTR Expression Resources

Authors: P.H. Thibodeau and P.J. Thomas

Department of Physiology

The University of Texas Southwestern Medical Center at Dallas

6001 Forest Park

Dallas, TX 75390-9040

email: philip.thomas@utsouthwestern.edu

 

INTRODUCTION

 

            The aim of this protocol is to accurately measure the affinity and stoichiometry

of nucleotide binding to an experimental sample.

 

MATERIALS

 

            1. Purified native (folded) protein

            2. Protein buffer (50mM Tris, 150mM NaCl, 5mM MgCl2, 1mM DTT pH 7.6)

            3. 6000 Ci/mmol αP32-ATP (MP Biomedicals)

            4. Protein buffer supplemented with Mg2+-ATP

            5. Ice cold protein buffer supplemented with 40 mM MgCl2

            6. Millipore HA 0.45 Ám nitrocellulose filters to fit manifold

            7. Filter manifold

            8. Test compounds

           

EXPERIMENTAL PROTOCOL

 

            First, prepare 10x ATP stocks with αP32-ATP, such that all concentrations of ATP can be readily counted by the scintillation equipment (this will vary by specific scintillation counter). Generally, one should try and maximize the specific activity of the ATP stocks as dilution and partial binding will decrease the signal in the experimental samples. Once the dilutions of ATP are made, count an aliquot from each dilution in the series. This will provide a reference for the dilution series later and allow one to calculate the specific activity in each of the samples. (The specific activity can also be calculated theoretically from the half-life and reference dates of the radioactivity.) Incubate protein and ATP at the desired temperature for sufficient time to reach equilibrium (up to 1 hr or more if necessary, depending on the binding kinetics). If the protein is sufficiently concentrated, small reaction volumes can be used which will aid in reducing the background noise of the experiment.  Total volume of 20 ml for each sample was used.  Buffer (either with or without test compounds 10mM final concentration) was pre equilibrated at 4oC.  Then protein was added to the buffer and incubated another 10 min at 4oC, and then ATP was added to the samples which were incubated for 60 minutes at 4oC.  During incubation assemble the filter manifold and pre-wet the filters with protein buffer. Apply vacuum to equilibrate the filters. Shut off the valve to the vacuum and apply the protein to the filters. Residual vacuum in the manifold will persist and aid in drawing the protein from the pipette tip onto the filter. Open the vacuum valve before washing.  Wash the filters 3 times with 1 ml ice cold buffer supplemented with MgCl2 with the vacuum applied constantly, do not allow the wash buffer to sit on the filters without vacuum. After the filters have been washed, turn off the vacuum, disassemble the manifold and count the radioactivity bound with scintillation equipment. Standard binding analyses can be performed using the collected data.

 

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