A dilution immunoassay to measure myosin regulatory light chain phosphorylation

We examined the quantitation of myosin regulatory light chain phosphorylati on (MRLCP) by Western blot and found both offset and saturation errors. The desirable characteristics of an MRLCP assay are that the dynamic range be 60- to 100-fold and that the detection threshold be known and preferably ve ry small relative to total MRLC concentration. No technique examined provid ed all these characteristics. However, accurate measurements can be obtaine d by including serial dilutions of the sample to provide a fractional calib ration scale in terms of the dephosphorylated light chain and by using inte rpolation of the phosphorylated band signal intensity to provide values for the relative phosphorylation ratio. We found that this method offers sever al advantages over methods that rely on signal ratios from single samples: The dilution ratio method is less subject to errors from differences in pro tein load, it offers estimates of the error in the individual measurement, and has some redundancy that increases the likelihood of obtaining a valid measurement despite gel or membrane artifacts. (C) 2000 Academic Press.