Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve.
Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.
Charette
Steve J
SJ
Institut de biologie intégrative et des systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Québec, Québec, Canada G1V 0A6. steve.charette@bcm.ulaval.ca
Lambert
Herman
H
Nadeau
Philippe J
PJ
Landry
Jacques
J
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western / Q000592:standards*; D002138:Calibration; D002460:Cell Line; D006801:Humans; D008163:Luminescent Measurements / Q000592:standards; D034622:RNA Interference; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human