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 µm 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.