Hi Sonam,
Could you please elaborate what you mean by "estimation of free glucose"? This protocol is based on acid-hydrolysis of glycogen in storage tissues, including liver and muscles, to produce free glucose that can be detected using the HK assay. The levels of glycogen could be low in fasted mice. However, according to what I could recall, we could always detect pretty good signals by the HK assay. Please make sure that the tissues are frozen in liquid nitrogen upon harvest, and it would be nice to prepare a glucose standard curve, just to to check the quality of the assay buffer.
Best,
Peichuan