Identifying new sperm Western blot loading controls

The measurement of protein expression level plays a pivotal role in both biological and medical studies. Housekeeping proteins, generally encoded by housekeeping genes are used as loading control proteins to normalize protein expression. Obviously, proper reference standards are essential for adequate analysis of protein expression. However, our study showed that the widely used normalisation proteins, whose expression levels varied greatly among sperm samples, were unsuitable for data standardisation. To uncover the proteins steadily expressed in sperm, we analysed several published transcriptome data of sperm. Seven proteins whose expression levels were relatively stable (co-efficient variation values less than 0.35) were selected and further evaluated by quantitative real-time polymerase chain reaction, Western Blot (WB) and immunocytochemistry. Our results showed that among the classical housekeeping proteins, only beta-tubulin remained constant in sperm samples from 85 individuals. Compared with other classical housekeeping proteins such as glyceraldehyde 3-phosphate dehydrogenase, actin and histone H3, Cullin-1 (CUL1) and F-box only protein 7 (FBXO7) seemed to be more suitable to be used as internal controls for WB in sperm protein studies. Combined with the locations of these proteins, CUL1 and FBXO7 were suggested to be used as a housekeeping protein for total proteins.