Quantitative Analysis of RadA Protein

Before transfer, total proteins loaded into the gel were detected by fluorescence with a stain-free imaging system (Bio-Rad). Intensity of protein bands was quantitated by Image Quant software and compared to a reference (WT) (36 (link)) to normalize data to total protein in each condition: normalization factor = total WT protein/total P15 protein. The stain-free blot was also analyzed to confirm the transfer quality for each condition. Then, the proteins visualized by chemiluminescence were quantitated to obtain the volume (intensity), and this volume was normalized as follows: normalization volume = volume × normalization factor. After normalization, the percentage of RadA protein was the result of the indicated volume/referenced volume (WT).