Protein quantification in SDS‑PAGE gels by densitometry using ImageJ/Fiji (with BSA normalization) v1

This protocol provides a detailed, step‑by‑step guide to quantify protein bands in stained SDS‑PAGE gels using ImageJ/Fiji. It explains how to prepare and scan the gel image, optimize it for analysis, and then quantify protein bands by two complementary approaches: Gel Analyzer method: generates lane profiles and integrates the peak areas under each band. Static ROI (Region of Interest) method: fixed rectangular or oval ROIs are drawn over each band and measured for mean gray value and integrated density. This approach provides a simpler, reproducible measurement when bands are regular and well separated. Both methods allow correction of background signal and can be combined with BSA protein standards for normalization or calibration (curve of Area vs ng). Results can then be exported to spreadsheets for statistical analysis and graphical representation. This protocol emphasizes the importance of consistent image processing, careful baseline setting, and use of proper controls (e.g., internal BSA reference or calibration standards). It also discusses troubleshooting (e.g., saturated bands, background correction, poor separation) and good practices for reporting. Estimated time: 45–75 min per gelLevel: Beginner–Intermediate