Selection and precautions of internal reference protein in western blot western experiment

Selection and precautions of internal reference genes in western blot western experiment

Western Blot experiments are commonly used to detect protein expression and the correctness of protein expression products. Compared with other methods for detecting proteins, the Western Blot experiment is simpler in qualitative and quantitative analysis of proteins and comparison with the amount of protein of interest.

Although, when it is smooth, 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 problems with antibodies and antibodies (the first antibody has problems or two) Anti-problem...) After all, as an active biomacromolecule, the reaction between antibodies and antigens is not as clear as 1+1, and this uncertain reagent is used to determine expression products that are rarely known, There is indeed some uncertainty. Therefore, a rigorous Western Blot experimental design requires a good reference system, which is very useful for the analysis of experimental results. Especially when there is a problem in the experiment, it is easy to find out the problem by means of the reference system, without having to scratch your head and blame.

A good reference system usually includes a molecular weight marker (used to determine the molecular weight corresponding to the protein band), a blank vector control (if the induction expression system should also have a pre-induction control), a positive control of a known amount of standard product, and There is an internal reference. 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. In the Western Blotting experiment , in addition to the steps of protein extraction, protein quantification, equal amount of protein loading, membrane transfection, target protein antibody incubation, color development, etc., internal reference detection is also required to correct protein quantification. The experimental error existing in the sample process ensures the accuracy of the experimental results.

In fact, the internal reference is the most easily overlooked item. 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 there is a basis for comparison. When the expression level is not high, the sample load is small. The difference is likely to affect the analysis of the results, so the internal parameters are needed. In published articles abroad, it has become a common practice to perform internal reference correction on the results of Western Blotting experiments. However, there are still many researchers in China who ignore the use of internal reference in the Western Blotting experiment, and use the protein concentration as the only method to standardize the sample loading between various samples that need to be compared with each other.

However, various protein concentration quantification methods have limitations and cannot completely determine the protein concentration of various samples. For direct quantification by UV method, it is suitable for testing relatively pure protein with relatively simple composition. Compared with colorimetric method, the operation is simple, but it is easily interfered by parallel substances such as DNA, and the sensitivity is low, and the concentration of protein is required to be high. Colorimetric determination of protein concentration generally has several methods 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 range of reducing agents that interfere with Lowry, BCA reactions (eg DTT, mercaptoethanol), but is still sensitive to detergents. The main disadvantage is that different standards can cause The results of the same sample are quite different and incomparable. In addition, an equal amount of loading is required for electrophoresis after protein quantification, and there is also an operational error in this step. The error in the quantification and loading steps can be easily corrected by using the internal reference in the Western blotting experiment.

The use of internal reference in Western Blotting is to additionally detect the internal reference with the antibody corresponding to the internal reference in 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, by means of detection The amount of sample internal reference can be used to correct the loading error, so that the semi-quantitative result is 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.

There are usually three methods for using internal parameters during the Western Blotting experiment.

The first is a super-convenient method of labeling internal reference, as long as the HRP-labeled internal reference antibody is added during incubation of the secondary antibody, as normal operation.

The second type is a common internal reference. When the molecular weight of the target protein is not much different from the molecular weight of the selected internal reference protein, the antibody of the target protein can be incubated for color development and detection, and then the antibody on the membrane is washed off using the Strip buffer. The antibody incubation and color detection of the internal reference protein were performed again.

Thirdly, when the molecular weight of the target protein is significantly different from the molecular weight of the selected internal reference protein, it can be pre-stained after the transfer, and the membrane is cut into two parts according to the size of the protein Marker, and the internal reference is made. The protein is separated from the target protein, and then the two membranes are incubated with the internal reference protein antibody and the antibody of the target protein, respectively, and the secondary antibody is incubated and developed.

Commonly used protein internal reference is GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and cytoskeletal protein beta-actin or beta-tubulin. Generally, the molecular weight of the protein of interest that we choose to participate in is preferably 5KD or more. So you need to know the molecular weight of the protein you are testing to choose the right internal reference!

Actin, actin, is an important backbone protein of cells. There are roughly six types of actin, four of which are specific for different muscle tissues, including alpha-skeletal muscle actin, alpha-cardiac muscle actin, alpha-smooth muscle actin, and gamma-smooth muscle actin. The other two are widely distributed. Among various tissues, including beta-actin (β-non-muscle) and gamma-non-muscle actin. The distribution of these different subtypes is different. There is little distribution of beta-actin in muscle tissue, and the myocardium is mainly alpha-cardiac muscle actin. Therefore, different organizations should have chosen different internal parameters, which cannot be generalized. Beta-actin is recognized as an internal reference for most tissues and cells. It is widely distributed in the cytoplasm and is abundantly expressed. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme involved in glycolysis, and tubulin is similar to actin and is a component of the cytoskeleton, but it is not a major component of muscle and should be a substitute. There are very few three simultaneous changes that require specific analysis. Once the above three internal parameters are changed at the same time, if the total protein can be replaced by the internal reference of the nucleus such as PCNA, TATA-box bingding protein (TBP) or even the internal parameters of mitochondria, of course, the probability of this possibility is negligible.

The expression of different isoforms has a great influence on the detection of proteins. Before buying antibodies, you should be familiar with your own target proteins. Many antibodies do not contain bands, which may not be a problem of antibody quality. Many companies' internal reference antibodies are prepared using antigenic fragments, and different companies choose different fragments. Take the most commonly used beta-actin as an example. Some companies choose the N-end and some choose the C-end. Preparation of antibodies (monoclonal antibody and polyclonal antibody) using N-terminal Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys 16aa as antigen Most of these antibodies are unable to detect actin bands in the myocardium and striated muscle. The main preparation of antibodies using C-terminal 16aa short peptides as antigens is Axxora PLATFORM (PSC-3777), EPITOMICS (1784-1, 1854-1, etc.), which can detect actin-positive signals in muscle cells. Sometimes the "tissue specificity" is produced when the internal reference is detected in the experiment, which is caused by the wrong choice of antibodies.

Several isomers of actin have tissue distribution specificity, and different types of actin have high sequence similarity (>90%). --actin is a skeletal protein distributed in non-muscle cells. When selected as β-actin, it is first ensured that it is widely expressed in tissues (especially when making tissue expression profiles of proteins). The antigen for the preparation of the β-actin antibody should then be an amino acid sequence identical to the other isomers of actin. The company prepares antibodies using a small short peptide of beta-actin as an immunogen. It may be because the selected short peptide is conserved between different types of actin, resulting in a certain tissue specificity of the prepared antibody. The short peptide is only present in the beta form, and the obtained antibody can only detect actin in non-muscle cells, and the selected short peptide is present in the six types, and the antibody can detect the cardiac, skeletal, smooth muscle, except muscle cells. The actin of the cell.

The selection of internal reference as a reference system in the Western Blot experiment is very illustrative of the experimental results. The selection of internal reference often uses some common housekeeping genes, and commonly used proteins include GAPDH and beta-actin. The selection of internal parameters in the Western Blot experiment and its use will directly affect the analysis of the experimental results.

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