I am currently engaged in a research project aimed at evaluating the expression and secretion of osteogenic differentiation markers in mesenchymal cells. My intention is to investigate transcription factors such as RUNX2, and proteins ALP and Osteocalcin as indicators of differentiation. However, I am facing a crucial decision: which method to employ to assess these markers accurately and reliably in my analyses. Considering the complexity and specificity of these markers, I am in pursuit of an approach that is as sensitive and specific as possible to detect even subtle variations in the expression of these proteins in total lysate, nuclear fraction, and cell supernatant. Therefore, I would like to inquire whether you could share your experiences and recommendations regarding the most appropriate methods for analyzing the expression and secretion of these osteogenic differentiation markers. I am open to suggestions.