Quantitative Westerns are performed in the same manner as qualitative Westerns, however the resulting signal must be captured and quantified. Digital capture and image analysis using the appropriate software allow for the sensitive quantitation of protein amounts. When not performed accurately, quantitative Western blots can give erroneous results care must be taken to understand the limits of the assay and the parameters that affect the ability to accurately measure protein quantity. Read below for factors that can affect quantitative Western blots. The process of Western blotting can be imprecise. Loading of samples, differences in transfer of protein to the membrane, antibody lots, etc. It is important to limit variability as much as possible.īe careful when loading samples onto the gel. Pipetting errors can affect the amount of protein in each lane.Īccurately time all incubations including equilibration of gel in transfer solution, blocking, antibody and substrate incubations.Keep transfer time standard to prevent transfer of proteins through the membrane or incomplete transfer from the gel.īuffer quality can affect transfer of proteins. Wet transfer provides a higher transfer quality than dry blot systems.ĭue to transfer and handling differences, only compare proteins on the same blot and not between blots. In the absence of appropriate endogenous references, lysates can be spiked with a known quantity of protein prior to loading.Make sure the internal reference does not vary with treatment conditions used in the experiment.Normalize the protein of interest to an internal reference, such as a “ housekeeping protein”.Normalization factors out inconsistencies in protein concentration resulting from loading errors or sample-to-sample variation. Sensitivity refers to the minimum amount of protein that can be detected and is dependent upon antibody quality, antibody concentration and exposure time. The linear dynamic range is the range in which signal intensity is proportional to the protein quantity on the blot. X-ray film can be saturated by signal and give false values.CCD has a higher sensitivity and broader linear dynamic.Use a charge-coupled device (CCD) camera-based imager Never use film If doing multiplexed Western, titrate each antibody individually prior to combining antibodies.ĭon’t use excessive amount of protein leading to saturation Use an antibody with a low limit of detection (LOD – amount of protein that can be distinguished from background).Use a highly sensitive antibody that allows precise quantitation of both strong and weak signals.Good quality quantitation of a Western blot occurs when the signal is within the linear range.
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