To test whether the expression product of a gene is correct, or to compare the relative changes in the amount of expression product, the preferred method is Western Blot. Although, when successful, Western Blot is very simple to do, but it is very annoying when it is not smooth. It can't produce results, false positives, and there are many strips in the end. Is there a problem with the primary antibody or the secondary antibody? The problem... After all, as an active biological macromolecule, the reaction between the antibody and the antigen is not as clear as 1+1, and the use of this uncertain reagent to determine the expression product, which is also poorly understood, is indeed There is a certain degree of uncertainty. Therefore, a rigorous Western Blot experimental design requires a good reference system, which is very useful for the analysis of experimental results.
We know that Western Blot should be used to compare the relative expression of the target protein under different conditions or in different tissues. The precondition is that the same amount of cells are loaded, and the basis for comparison is obtained. When the special expression is not high, the difference in the amount of the sample is likely to affect the analysis of the results. Amy Jie recommends using internal reference antibodies to test your experimental system or to calibrate your results. The internal reference is the Internal Control. For mammalian cell expression, it is generally referred to as Housekeeping Proteins, which are expressed relatively constant in tissues and cells, and the expression level of the protein is detected. It is often used as a reference when changing. The use of internal reference in Western Blotting is actually the use of the internal antibody corresponding to the internal reference in the WB process to detect the internal reference, so that the expression of the internal reference can be detected while detecting the target product, because the expression of the internal reference in each tissue and cell is relatively constant, by means of detection 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 condition is complete, the entire Western Blot color development or 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 test is performed, and the internal parameters and the amount of the target protein are separately detected. The amount of protein in each sample was divided by the content of the internal reference, and the obtained value was the relative content of the target protein in each sample after the internal reference correction, and then the value was compared and analyzed between the samples to obtain the target protein content between different samples. The actual change results. If the sample volume is sufficient, the internal reference can be detected first, and the internal color bands of the samples are observed to be consistent. The sample volume of each sample is adjusted according to the difference size, and the Western Blotting experiment is performed again until the internal parameters are consistent; if the internal parameters are consistent, the Analysis of changes in protein expression of proteins between different samples. This is a little troublesome, but it can guarantee that the results are more convincing and more credible. After all, our experiment is a rigorous job.
We know that Western Blot should be used to compare the relative expression of the target protein under different conditions or in different tissues. The precondition is that the same amount of cells are loaded, and the basis for comparison is obtained. When the special expression is not high, the difference in the amount of the sample is likely to affect the analysis of the results. Amy Jie recommends using internal reference antibodies to test your experimental system or to calibrate your results. The internal reference is the Internal Control. For mammalian cell expression, it is generally referred to as Housekeeping Proteins, which are expressed relatively constant in tissues and cells, and the expression level of the protein is detected. It is often used as a reference when changing. The use of internal reference in Western Blotting is actually the use of the internal antibody corresponding to the internal reference in the WB process to detect the internal reference, so that the expression of the internal reference can be detected while detecting the target product, because the expression of the internal reference in each tissue and cell is relatively constant, by means of detection 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 condition is complete, the entire Western Blot color development or 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 test is performed, and the internal parameters and the amount of the target protein are separately detected. The amount of protein in each sample was divided by the content of the internal reference, and the obtained value was the relative content of the target protein in each sample after the internal reference correction, and then the value was compared and analyzed between the samples to obtain the target protein content between different samples. The actual change results. If the sample volume is sufficient, the internal reference can be detected first, and the internal color bands of the samples are observed to be consistent. The sample volume of each sample is adjusted according to the difference size, and the Western Blotting experiment is performed again until the internal parameters are consistent; if the internal parameters are consistent, the Analysis of changes in protein expression of proteins between different samples. This is a little troublesome, but it can guarantee that the results are more convincing and more credible. After all, our experiment is a rigorous job.
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