What You Need to Konw in Western Blot Experiments

Western Blot, also known as western blot analysis and immunoblotting, is a technique widely used in the detection and analysis of proteins. It uses polyacrylamide gel electrophoresis. The detected substance is protein, and the “probe” is an antibody. “Use a labeled secondary antibody. Western blot analysis is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. Medicilon Biopharmaceuticals can provide Western blotting experimental technology services

The working principle of western blot analysis: the protein sample separated by PAGE is transferred to a solid-phase carrier (such as nitrocellulose membrane). The solid-phase carrier adsorbs the protein in the form of non-covalent bonds, and can maintain the types and types of peptides separated by electrophoresis. Its biological activity remains unchanged. The protein or polypeptide on the solid-phase carrier is used as the antigen to react with the corresponding antibody, then react with the enzyme-labeled secondary antibody, and detect the protein expressed by the specific target gene separated by electrophoresis through the substrate color. It is also widely used to detect the expression of protein levels.

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About protein extraction:

(1) Cracking buffer:

Different lysis buffers have different uses. In addition to the lysis buffer, various protease inhibitors and phosphatase inhibitors are necessary to prevent protein degradation and dephosphorylation; when extracting proteins, the lysis solution alone cannot achieve complete extraction. If possible, the extraction suspension can be sonicated for 10-20s; in addition, if there is no lysis buffer, 1×SDSloading buffer can be used instead to extract the protein. The effect is similar to that of SDS lysis solution, but The extracted sample cannot be measured for concentration.

(2) Interfering protein:

There are serum components in cultured cells and animal tissues. There are a large number of albumin and immunoglobulins in serum. These proteins will often interfere with bands. Generally, albumin bands are at 70kDa, and immunoglobulin bands appear at 50kDa. Place. When the content of a certain protein exceeds a certain amount, even a small amount of non-specific adsorption may cause signal enrichment and cause miscellaneous bands. Therefore, when extracting cell proteins, the cells must be rinsed at least 3 times with PBS to remove Residual medium components. When extracting the tissue, try to remove the blood residue in the tissue, so that the interference factors in the sample will be minimized.

The choice of gel concentration and electrophoresis conditions:

The correct choice of gel concentration and electrophoresis conditions can maximize the separation of the target protein region, so as to minimize the influence of some factors such as the position of the band and the position of the mixed band.

Conditions and precautions for film transfer:

(1) Transfer film conditions:

For indicators with a molecular weight of 10-15kDa, the transfer time should not be too long. 100V and ice bath for 45min are sufficient; for indicators with a molecular weight of 150kDa or more, 100V and ice bath for 2h are also sufficient; other indicators between 15-150kDa are 100V , 1h ice bath can fully guarantee the effect.

(2) Film transfer operation:

The size of the prepared PVDF membrane should be the same or slightly smaller than the size of the cut gel, and the size of the filter paper and sponge should also be the same. The “sandwich” should not be too thick or too thin. Too thick will easily deform the glue, the strip will bend, and too thin bubbles will easily enter, resulting in incomplete strips. These can be improved by increasing and reducing the amount of filter paper; the purpose must be improved. The protein molecular weight region is attached to the middle part of the membrane. Distinguish the positive and negative electrodes clearly and avoid transferring in the opposite direction. The transfer buffer should be pre-cooled in advance, and the entire transfer process also needs to be performed in an ice bath.

The difference between actual and predicted results of molecular weight:

Factors that may cause the predicted and actual molecular weight to be different:

(1) Error of pre-stained marker:

Since the pre-stained marker is formed by coupling a standard protein and a colored dye, the surface structure of the coupled protein is not as good as the linear relationship of the natural protein due to the change of the surface structure; Different batches, the molecular weight of each band for each batch will vary slightly, so when using pre-stained markers, a 5-10% molecular weight error is unavoidable. If the exact molecular weight is required Use non-pre-stained markers for calibration.

(2) Species difference:

Many proteins have different lengths in human, rat, and mouse sources, and specific problems need to be analyzed in detail.

(3) The nature of the protein itself:

The molecular weight value is only a relative value accumulated by an amino acid value, which is different from the actual value itself;

The presence of post-translational modifications such as phosphorylation, acetylation, and glycosylation of proteins can lead to an increase in molecular weight. For example, the molecular weight of common CD family protein glycosylation modifications will increase a lot;

The protein has a post-translational enzyme precursor state and an activated cleavage state, which can make the molecular weight smaller. For example, the size of the enzyme source and the activated state of the MMP2 protein are 72kDa and 63kDa respectively;

There are multiple transcripts of proteins that are encoded by one gene. The molecular weights of proteins translated from different transcripts are different. For example, the protein of JNK is divided into three subtypes, JNK1, JNK2, and JNK3, and the homology of the three subtypes. High, and there are transcripts of different molecular weights for each subtype; to correctly grasp the protein content, not only read the instructions, but also pay attention to the relevant background knowledge of the protein.

(4) Uniprot:

This database provides some references on protein predicted molecular weight, subtype classification, transcript type and the initial discovery of this protein.

About “whiteboard” and “blackboard”:

Western results are often in a certain condition between “whiteboard” and “blackboard”:

(1) White board: The meaning of “white board” is that certain conditions are insufficient: first, consider the problem of protein extraction, whether the sample concentration is too low, a normal Western experiment requires about 20-40μg protein per well; the dilution ratio of the primary antibody is generally Novices will do WB according to the ratio of the manual. Generally, the manual is between 1:500-2000. If it is a whiteboard, the antibody ratio needs to be increased. If the minimum recommended ratio of the manual is still a “whiteboard”, it should not be The dilution ratio is a problem; the primary antibody incubation time, the conventional primary antibody incubation condition is 4 overnight, sometimes the antibody response is “sluggish” and may require a more intense incubation time, such as extending the incubation time to 2 days, or placing a shaker Incubate overnight in the refrigerator with slow shaking. We do not recommend 37 for antibody incubation; the choice of color development system, generally speaking, ECL chemiluminescence is more than 5-10 times more sensitive than DAB, so try to use ECL; ECL luminescent solution is also divided into many types. We have tried the normal, sensitive, and ultra-sensitive types, and there are indeed very obvious differences in the signal; of course, extending the exposure time is also the most common way to improve the signal sensitivity.

(2) Blackboard: As the name implies, “blackboard” means that some conditions are overdone. Compared with “whiteboard”, the meaning of “blackboard” is to consider the amount of sample; reduce the dilution ratio of the primary antibody; reduce the dilution ratio of the secondary antibody. Very mature product, the quality is guaranteed, we have been using HRP secondary antibody, 1:5000, 40min at room temperature, basically do not need to explore the conditions of the secondary antibody, in summary, it is still necessary to buy the first antibody and the second antibody from the reliable manufacturer; shorten the exposure time.

About closure

Sealing is also a key link in Western. The general condition is that 5% skimmed milk powder is dissolved in 1×TBST. The shaker is slowly shaken at room temperature and sealed for 1 hour. In addition to this, there is another blocking solution—0.5% gelatin. Both of these two blocking solutions are suitable for conventional Western experiments. The trade-off relationship between them can be approximated. If the result of blocking with skimmed milk powder is “whiteboard” or the signal is weak, you can consider changing to gelatin. If you use gelatin, the result There are many miscellaneous belts, and the background is dark. You can consider using skimmed milk powder; sometimes the two can be mixed and used.

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