Immunoprecipitation-Coupled In Vitro Kinase Assay Measuring c-SRC Catalytic Activity After Dasatinib Treatment in HCT116 Colorectal Cancer Cells

Two arms (0.1% DMSO vs 100 nM dasatinib, 1 h) plus a dilution sub-series (0, 1, 10, 100 nM dasatinib) for a partial dose-response, each n=4 independent biological replicates. Each kinase reaction is run in technical duplicate. A heat-inactivated SRC IP and a no-substrate reaction define assay background. Plate layout for the luminescent ADP/ATP readout is randomized; the operator reading luminescence is blinded to well identity.

BSL-2 for HCT116 human tumor cells; biosafety cabinet for cell handling. Sodium orthovanadate (Na3VO4) is toxic—handle with gloves, avoid inhalation of powder, neutralize before disposal. Dasatinib and DMSO handled with nitrile gloves. ADP-Glo reagents are low-hazard but follow SDS. Cell waste decontaminated in 10% bleach 30 min; chemical waste segregated (vanadate as heavy-metal waste). Use white opaque plates and limit ambient light before luminescence reads.

Negative controls: isotype IgG IP (no specific kinase), heat-inactivated SRC IP, and no-substrate reaction (defines background ADP). Positive control: vehicle (DMSO) IP showing full activity, and optionally recombinant active SRC as an assay-performance standard. The captured-SRC immunoblot is an internal normalization control ensuring equal enzyme input. A no-ATP reaction confirms signal is ATP-dependent.

Vehicle SRC produces robust ADP-Glo luminescence well above heat-inactivated and IgG backgrounds (signal-to-background >10). Dasatinib reduces activity dose-dependently with >80% inhibition at 100 nM and an apparent cellular IC50 in the low-nanomolar range. Captured-SRC immunoblot signal is equivalent across arms (<20% variation), confirming catalytic rather than expression-level change.

• HCT116 cells (ATCC CCL-247), colorectal carcinoma
• McCoy's 5A medium + 10% FBS + 1% pen/strep
• Dasatinib (BMS-354825), 1 mM stock in DMSO
• Native IP lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM Na3VO4)
• Halt protease + phosphatase inhibitors (100x)
• Anti-SRC (36D10) rabbit mAb (IP-grade), 3 µg/IP
• Normal rabbit IgG isotype control, 3 µg/IP
• Protein A/G magnetic beads
• Kinase reaction buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA)
• SRC substrate peptide (e.g., AEEEIYGEFEAKKKK), 0.2 mM
• ATP, 10 µM final
• ADP-Glo luminescent kinase activity kit
• Anti-SRC blotting antibody for IP normalization, 1:1000

To immunoprecipitate endogenous c-SRC from HCT116 lysates and assay its intrinsic kinase activity in vitro against a synthetic SRC-optimal peptide substrate, quantifying ATP consumption (or phosphopeptide product) to compare DMSO- versus dasatinib-treated cells. The assay isolates catalytic activity from expression level, providing a direct functional pharmacodynamic readout of SRC inhibition.

1. Grow HCT116 in 10-cm dishes to 80%; treat 1 h with 0.1% DMSO or dasatinib (1-100 nM).
2. Wash 2x ice-cold PBS; lyse in 0.8 mL IP buffer + inhibitors, 20 min on ice.
3. Centrifuge 16,000 x g, 15 min, 4 °C; quantify by BCA; use 1 mg per IP.
4. Pre-clear with 25 µL beads 30 min; add 3 µg anti-SRC; rotate 3 h at 4 °C.
5. Add 30 µL beads, rotate 1 h; wash 3x lysis buffer, then 2x kinase buffer (removes vanadate/ATP carryover).
6. Resuspend bead-bound SRC in 25 µL kinase buffer; split into duplicate 12.5 µL reactions.
7. Initiate with peptide substrate (0.2 mM) + ATP (10 µM final); incubate 30 min at 30 °C, shaking.
8. Run a parallel heat-inactivated IP (95 °C, 5 min) and a no-peptide reaction as backgrounds.
9. Transfer reactions to a white 384-well plate; add ADP-Glo reagent per kit, then kinase detection reagent.
10. Read luminescence after 30 min on a plate reader.
11. Save a bead aliquot for anti-SRC immunoblot to normalize activity to captured SRC.

Independent variable: dasatinib concentration (0-100 nM). Dependent variable: SRC kinase activity (luminescence-derived ADP signal) normalized to immunoprecipitated SRC protein. Controlled variables: IP input mass (1 mg), antibody amount (3 µg), ATP (10 µM) and peptide (0.2 mM) concentrations, reaction time (30 min) and temperature (30 °C), and wash regimen to remove cellular ATP/inhibitor carryover.

We hypothesize that dasatinib, an ATP-competitive SRC/ABL inhibitor, treats cells such that immunoprecipitated SRC retains the inhibitor in its ATP pocket and exhibits markedly reduced in vitro kinase activity (>80% reduction at 100 nM) relative to vehicle, while total immunoprecipitated SRC protein is unchanged, demonstrating activity loss is catalytic rather than expression-driven.

Subtract the no-substrate/heat-inactivated background from each reaction's luminescence, average technical duplicates, then divide by the densitometric captured-SRC signal to yield specific activity. Express each as a percentage of the within-replicate vehicle. Fit percent activity vs log[dasatinib] to a four-parameter logistic in GraphPad Prism to estimate cellular IC50. Convert luminescence to ADP using an ATP-to-ADP standard curve if absolute kinetics are required.

1. Low overall activity: ensure the second kinase-buffer wash fully removed EDTA/vanadate that inhibit the in vitro reaction; verify MgCl2 in reaction buffer. 2. High background luminescence: include no-substrate control and ensure complete ATP removal during washes. 3. No dasatinib effect: inhibitor may have washed out during bead washing—dasatinib is ATP-competitive and reversible, so keep wash time short/cold and consider re-adding inhibitor to reactions. 4. Variable replicates: normalize strictly to captured-SRC immunoblot. 5. Saturated luminescence: shorten incubation or dilute beads; saturation breaks the linear ATP-to-ADP relationship.

n=4 biological replicates, technical duplicates averaged. Compare normalized SRC activity across concentrations by one-way ANOVA with Dunnett's correction vs vehicle (alpha = 0.05). Report IC50 with 95% CI from the fit. Power analysis (G*Power, one-way ANOVA, large effect f≈1.0, alpha 0.05, power 0.8) supports n=4 across four groups; report exact p-values and eta-squared effect sizes.