Total Protein Normalization: The Better Alternative Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". Total protein normalization of HKPs yields consistent band intensities. Immunodetection measurements of housekeeping protein levels show poor linearity and do not accurately indicate cell lysate loading levels. The expression level of housekeeping proteins can change due to:ĭifferences in five candidate housekeeping proteins and total protein staining between tumor and non-cancerous tissues in the validation sample set. Variation in Housekeeping Protein (HKP) Expression Levels Furthermore, HKP expression levels may not be constant but instead vary with different experimental treatments and other factors. Thus, to detect the target protein of interest, large amounts of cell lysate may need to be loaded, resulting in overloading of HKPs, yielding oversaturated reference bands, out of their linear range. Unfortunately, housekeeping proteins are usually highly expressed, whereas target proteins are often expressed only in low abundance. Reliable assessment of the changes in target protein expression levels requires the measurement of both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Typically, a "housekeeping protein" (HKP) such as β-actin, β-tubulin, or GAPDH is used as a loading control, with the assumption that the expression levels of these proteins remains constant. Proper western blot normalization is required to show that the changes in band intensities correlate to the biological changes in your samples.Ĭonventional loading controls rely on consistent levels of a reference protein in each sample. Western blot normalization allows you to faithfully compare changes in protein expression by establishing the baseline needed to correct against common errors such as inconsistent sample preparation, pipetting error, and uneven protein transfer. ![]() Common blot-to-blot variations are accurately assessed by an internal loading control.All quantified targets' signal intensities fall within the linear range of the imaging system.The amount of protein loaded is proportional to the signal intensity.A quantitative western blot accurately measures changes in protein expression by making relative comparisons between different treatments.Ī properly designed quantitative western blotting experiment ensures that:
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |