We investigated the effects of tissue samples taken from rat brain on the reliability of three protein quantification kits: the Bradford assay, the 2-D Quant Kit, and the EZQ Protein Quantitation Kit. All three assays measured significantly smaller amounts of protein after extraction than the reference values before extraction. Only small effects were seen in homogenates, but very pronounced differences in membrane-enriched and highly lipophilic subcellular fractions. Researchers should evaluate which method of protein quantification is best qualified for their specific experimental design.