Internal parameter setting in Western Blot

Internal parameter setting in Western Blot

Western Blot (WB) is the preferred method to detect the correct expression of a gene and the amount of expression. WB is easy to operate, it can not only analyze the expression product qualitatively, but also indicate the relative change of the target protein amount. As an active biological macromolecule, the reaction between antibodies and antigens is relatively complicated, so there are certain uncertainties when used to determine the expression products. Therefore, adding a good reference system to the WB experimental design is very helpful for the analysis of the experimental results. The reference system usually includes a molecular weight marker (used to determine the molecular weight corresponding to the protein band), a blank vector control (if it is an induced expression system, there should be a control before induction), a positive control with a known amount of standard product, and internal controls .

Internal control is the most easily overlooked item. If WB is used to compare the relative expression of the target protein under different conditions or in different tissues, the same amount of cells can be used for sample comparison. In particular, when the expression level is not high, the difference in the sample volume is likely to affect the analysis of the results, so the internal reference is particularly important at this time.

Internal control is internal control. For mammalian cell expression, it generally refers to the protein encoded by housekeeping genes (Housekeeping Proteins). Their expression in various tissues and cells is relatively constant. It is often used as a reference when changing. In the WB experiment, in addition to the steps of protein extraction, protein quantification, electrophoresis of equal protein loading, membrane transfer, target protein antibody incubation, and color development, internal reference detection is also required to correct protein quantification and sample loading The experimental errors in the process ensure the accuracy of the experimental results.

The determination of protein concentration is the only way to compare the sample load between various samples. However, various methods of protein concentration quantification have limitations, and the exact protein concentration of various samples cannot be determined completely and accurately. For example, the UV method is directly quantified, which is suitable for testing relatively pure proteins with relatively single components. Compared with the colorimetric method, the operation is simple, but it is susceptible to interference by parallel substances, such as DNA; and the sensitivity is low, requiring the concentration of protein Higher. There are several methods for colorimetric determination of protein concentration, such as BCA, Bradford and Lowry. Both the BCA method and the Lowry method are susceptible to interference between proteins and detergents. The Bradford method is the most sensitive and compatible with a series of reducing agents (such as DTT, mercaptoethanol) that interfere with the Lowry and BCA reactions. However, it is still sensitive to detergents, and its main disadvantage is that different standards will cause the results of the same sample to be very different and incomparable. In addition, when quantifying the protein after electrophoresis, an equal amount of sample loading is required, and there is an operation error in this step. Using the internal reference in the WB experiment, you can easily correct the errors caused by the quantification and loading steps.

The use of internal reference in WB is actually the detection of the internal reference with the antibody corresponding to the internal reference during the WB process, so that the expression of the internal reference can be detected while detecting the target product. Since the expression of the internal reference in each tissue and cell is relatively constant, with the help of detecting each The amount of internal reference in each sample can be used to correct the loading error, so that the semi-quantitative results are more reliable. In addition, the internal reference can be used as a blank control to detect whether the protein transfer is complete, whether the entire WB color is developed, or whether the luminescence system is normal.

The analysis of the experimental results is actually very simple: if the amount of protein in the sample is limited, only one electrophoresis transfer membrane experiment can be performed to detect the internal parameters of the sample and the amount of target protein separately. Divide the target protein amount of each sample by its internal reference content, and the value obtained is the relative content of the target protein in each sample after internal reference correction. Then use this value for comparison and analysis between samples to obtain the target protein content between different samples. The actual change results. If the sample volume is sufficient, you can check the internal parameters first to observe whether the internal reference color bands are consistent between samples. Adjust the sample loading of each sample according to the size of the difference and repeat the Western Blotting experiment until the internal parameters are consistent; Analysis of changes in target protein expression between different samples. Commonly used protein internal controls include GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and cytoskeletal protein beta-actin or beta-tubulin.