Table 3.

Comparison of western blotting with the combined method of IF and fluorescence densitometry

	WESTERN BLOTTING	IF + FLUORESCENCE DENSITOMETRY
Detection method	Antigen–antibody reaction	Antigen–antibody reaction
Relative or absolute quantification	Possible	Possible (e.g., number of puncta)
Size of protein	Can be determined	Cannot be determined
Sample size	10 μg total protein. May limit detection of protein at very low concentration.	Sections or cells
Sensitivity	Very sensitive (enzyme-conjugated detection), but membrane transfer may limit detection.	Very sensitive
Optimizations needed	High	High
Detection of multiple proteins	Yes, in same gel	Yes
Localization of protein in cells/tissues	No	Yes
Subcellular localization of protein	No	Yes, subcellular quantification is possible.
Colocalization with other proteins or mRNA	Not possible	Possible
Tolerate partially degraded protein	Possible	Yes
Time consumption	Work intensive	Less work intensive