Enhanced protein extraction and quantification protocol for microsamples: An ultra‐sensitive workflow for low‐volume, low‐concentration total protein lysates

Accurate protein quantification at low concentrations and small volumes is critical for advancing small-scale research, such as microvasculature studies. However, existing microscale protocols often require ≥5 μL of sample or highly concentrated lysates, limiting their applicability in contexts with scarce material. To overcome these limitations, we developed the Nano-Extraction BCA-Optimized Workflow (NEBOW), a novel method requiring only 2 μL of sample and capable of detecting protein concentrations as low as 0.01 mg/mL. Optimized for the NanoDrop™ One UV-Vis Spectrophotometer, this workflow demonstrated significantly enhanced sensitivity and reproducibility compared to the standard BCA assay. Paired t tests (p < 0.01) and TOST equivalence testing (15% margin) confirmed key differences, with the NEBOW method producing steeper standard curves and more consistent results at low concentrations. Bland-Altman analysis showed that standard BCA tends to overestimate protein levels, while the NEBOW method maintained accuracy across a range of low-input samples. Western blot validation supported the improved performance of the new workflow. This approach offers a reliable, cost-effective solution for protein quantification when sample availability is limited, without sacrificing accuracy or sample integrity.