3.3 Quantitative
AlphaScreen Assay
to Measure Soluble
Target Protein
for CETSA
1. Dilute 0.6 mL of 5Â SureFire Lysis Buffer in 2.4 mL of dH 2 O
to give 3 mL of 1Â SureFire Lysis Buffer. Harvest cells (see
Note 10) and pellet before resuspending in 1Â SureFire Lysis
Buffer to a final cell density of 1 Â 10
7 cells/mL. Incubate for
1–2 h at room temperature or >2 h at 4
C to ensure homogeneous cell lysis (see Note 11).
2. Dispense 3 μL/well of cell lysate into an AlphaScreen compatible white, shallow 384 well microplate, e.g., ProxiPlate384 Plus.
3. Dilute 5 mL of 10Â ImmunoAssay Buffer in 45 mL of dH 2 O
to give 50 mL of 1Â ImmunoAssay Buffer. Prepare microcentrifuge tubes with 500 μL of 1Â ImmunoAssay Buffer. To each
tube, add 0.5 μL (1:1000) of one mouse-derived anti-target
antibody and 0.5 μL (1:1000) of one rabbit-derived anti-target
antibody, prepared in a matrix such that each mouse-derived
antibody is tested in combination with each rabbit-derived
antibody. Include a buffer only control.
4. Dispense 3 μL/well of each antibody combination into separate wells of cell lysate in the ProxiPlate-384 plate, ensure
mixing by brief centrifugation at 300 Â g for 10 s, and then
seal the plate and incubate for 1 h at room temperature.
5. Dilute 0.3 mL of 10Â ImmunoAssay Buffer in 2.7 mL of
dH 2 O to give 3 mL of 1Â ImmunoAssay Buffer. Under subdued light, add 72 μL of anti-Mouse IgG Alpha Donor beads
to give a concentration of 120 μg/mL and 18 μL of anti-rabbit
IgG (Fc specific) AlphaLISA Acceptor beads to give a concentration of 30 μg/mL. Dispense 3 μL/well of AlphaScreen
reagents onto the cell lysates and antibody mix in the
ProxiPlate-384, ensure mixing by brief centrifugation at
300 Â g for 10 s, and then seal the plate and incubate for
4–16 h at room temperature.
6. Analyze AlphaScreen signal using a suitable plate reader, e.g.,
EnVision. Excite at 680 nm for 180 ms and measure emission
at 570 nm for 550 ms.
7. For the antibody pair(s) that give AlphaScreen signal above the
buffer control in step 6, repeat steps 2–6 with variations in the
dilution of the mouse-derived and rabbit-derived antibody and
the cell density of the lysate to achieve a maximum AlphaScreen
signal, which falls within a linear range with respect to cell
number (see Note 12). Dilutions of the mouse antibody at
1:200, 1:500, 1:1000, 1:2000, 1:5000, and 1:10,000 should
be titrated against the same dilutions of the rabbit antibody.
Once optimal antibody dilutions have been established, cell
density should be titrated from 2 Â 10
7 cells/mL to
2 Â 10
6 cells/mL (see Note 13).
208
Joseph Shaw and Christopher Stubbs
AlphaScreen Assay
to Measure Soluble
Target Protein
for CETSA
1. Dilute 0.6 mL of 5Â SureFire Lysis Buffer in 2.4 mL of dH 2 O
to give 3 mL of 1Â SureFire Lysis Buffer. Harvest cells (see
Note 10) and pellet before resuspending in 1Â SureFire Lysis
Buffer to a final cell density of 1 Â 10
7 cells/mL. Incubate for
1–2 h at room temperature or >2 h at 4
C to ensure homogeneous cell lysis (see Note 11).
2. Dispense 3 μL/well of cell lysate into an AlphaScreen compatible white, shallow 384 well microplate, e.g., ProxiPlate384 Plus.
3. Dilute 5 mL of 10Â ImmunoAssay Buffer in 45 mL of dH 2 O
to give 50 mL of 1Â ImmunoAssay Buffer. Prepare microcentrifuge tubes with 500 μL of 1Â ImmunoAssay Buffer. To each
tube, add 0.5 μL (1:1000) of one mouse-derived anti-target
antibody and 0.5 μL (1:1000) of one rabbit-derived anti-target
antibody, prepared in a matrix such that each mouse-derived
antibody is tested in combination with each rabbit-derived
antibody. Include a buffer only control.
4. Dispense 3 μL/well of each antibody combination into separate wells of cell lysate in the ProxiPlate-384 plate, ensure
mixing by brief centrifugation at 300 Â g for 10 s, and then
seal the plate and incubate for 1 h at room temperature.
5. Dilute 0.3 mL of 10Â ImmunoAssay Buffer in 2.7 mL of
dH 2 O to give 3 mL of 1Â ImmunoAssay Buffer. Under subdued light, add 72 μL of anti-Mouse IgG Alpha Donor beads
to give a concentration of 120 μg/mL and 18 μL of anti-rabbit
IgG (Fc specific) AlphaLISA Acceptor beads to give a concentration of 30 μg/mL. Dispense 3 μL/well of AlphaScreen
reagents onto the cell lysates and antibody mix in the
ProxiPlate-384, ensure mixing by brief centrifugation at
300 Â g for 10 s, and then seal the plate and incubate for
4–16 h at room temperature.
6. Analyze AlphaScreen signal using a suitable plate reader, e.g.,
EnVision. Excite at 680 nm for 180 ms and measure emission
at 570 nm for 550 ms.
7. For the antibody pair(s) that give AlphaScreen signal above the
buffer control in step 6, repeat steps 2–6 with variations in the
dilution of the mouse-derived and rabbit-derived antibody and
the cell density of the lysate to achieve a maximum AlphaScreen
signal, which falls within a linear range with respect to cell
number (see Note 12). Dilutions of the mouse antibody at
1:200, 1:500, 1:1000, 1:2000, 1:5000, and 1:10,000 should
be titrated against the same dilutions of the rabbit antibody.
Once optimal antibody dilutions have been established, cell
density should be titrated from 2 Â 10
7 cells/mL to
2 Â 10
6 cells/mL (see Note 13).
208
Joseph Shaw and Christopher Stubbs