Response to the Comment "On Determination of Force on Bead-Surface Linker Proteins" by Rasool Dezhkam and Cheng Zhu (smll.202510363).

A recently published comment challenges the data analysis we employed to quantify nanomechanical properties in our force spectroscopy experiments. In the corresponding measurements, sub-µm-sized fluorescent beads are linked via nm-sized macromolecules ("linker") to the bottom of a microfluidic channel. Application of a flow through the channel generates a well-defined shear force acting on the bead, which deforms the linker and can be followed with nm spatial and sub-pN force resolution and high data throughput using optical microscopy. The comment challenges our analysis and proposes a different model for the analysis of our data. Based on this model, the authors claim that for certain measurements, the loading force was underestimated by a factor of up to 30. In our response, we discuss the rationale behind our model in detail and provide experimental evidence that supports our rationale. This data shows that the proposed model is oversimplified in the sense that important restoring forces have not been taken into account. As a consequence, the proposed model does not reflect all features of our data and is therefore not applicable to our experimental setting. We therefore dismiss the claim that we underestimated the loading force by orders of magnitude.