Western Blot uses polyacrylamide gel electrophoresis. Protein samples separated by PAGE are transferred to a solid phase carrier. The solid phase carrier adsorbs proteins in a non-covalent bond and maintains the type of electrophoretic separation and its biological activity. constant. The protein or polypeptide on the solid phase carrier is used as an antigen, and the corresponding antibody is immunoreacted, and then reacted with the labeled second antibody, and the specific target gene expression separated by electrophoresis is detected by substrate color development or autoradiography. Protein composition. This technique is also widely used to detect expression at the protein level.
With regard to Western Blot technology, our laboratory has accumulated several experiences based on years of experience:
1. Antibody selection
For most laboratories in China, it is a headache to do western blot experiments. The reason is very simple. Buying imported antibodies is stretched and it takes a lot of courage to buy domestic antibodies. In the laboratory western blot experiment, we used imported antibodies, imported antibodies domestically packaged, domestic antibodies, the quality is not good. Imported antibodies generally do not appear to be lost: Abcam varieties are all, the quality is excellent, but the price is high; Sigma is the most expensive; the more cost-effective is the CST antibody, the shortage is not the CST variety. Santa's polyclonal antibody is of a good quality, but Santa's monoclonal antibody is still at risk. It is estimated that this is the industry consensus.
We also used a lot of imported antibody domestic packaging, probably about 50% of the antibody. If it can be made, there is also a problem, that is, most of the antibodies can only be used once for domestic antibodies, but The quality is really not flattering. Western blot can be made very little, and there are many miscellaneous bands and the background is not clean.
2. Western Blot experimental conditions
The specific reagent formula is prepared by the application of the transfer electrophoresis buffer in the appendix of the BioReagents book. We are 300 mAh / 2 hours of film transfer. The film must be made into an ice water bath, which is to soak the tank in the ice water mixture.
3. About milk powder, film selection
Milk powder seems simple, but it is very important. If you want to make it, you can go to the supermarket to buy skim milk powder. The quality of the milk powder is not good, and it will eventually lead to development or darkness, or nothing. The BD we are using now is very stable and recyclable. We have used NC film and PVDF film. The advantage of PVDF film is that the protein load is large and the toughness is good. The only trouble is to use methanol for a few minutes beforehand in order to enhance the positive charge of the membrane. After the methanol is bubbled, it will be soaked in the transfer buffer for one or two minutes.
4. About the coloring solution
We use imported high-sensitivity ECL coloring solution. It is generally no problem to detect the internal reference with an ordinary color developing solution, but it is not easy to develop a protein with a low expression level, and the hypersensitivity is different, even if the protein expression is low, it can be detected.
5. The key to Western Blot experiment
Western Blot has many operation steps, and each step of the mistake will cause a total failure. In general, antibodies are still the key to success or failure. If the antibodies are not good, the gods are not recruited. Among them, the internal reference antibody is very important, because the selection of the internal reference antibody is related to the evaluation of the whole experiment. Actin, Tubulin, GAPDH we have used, Actin, Tubulin's shortcomings are sometimes doubling, more stable is GAPDH. If you look carefully at the articles on cell, nature, and science, most of them use GAPDH as an internal reference. Imported, domestically packaged, domestically produced, we have used it, the effect is ok.
From the western experiment, we conclude that the experiment can not save money, and a bad link will lead to a total failure. The same is true for experimental design, and the negligence of one step leads to a logical confusion between the entire experiment and the evaluation. If you want to pursue cost-effectiveness, that is, how to maximize the benefits of the poor Rolls-Royce, then from the very beginning, you should choose the reagents according to the principle of accounting after the fall, and you should count the general ledger; if you want to make the experiment smooth, you have to pay attention to each one. The link is step by step, and the details determine success or failure. Until now, as long as the antibody works, there are almost no molecules that we can't do, and Western gives us endless confidence.
Related Links:
- Western Blotting (Western blot) technology
- Co-immunoprecipitation (CO-IP)
- Protein two-dimensional electrophoresis (2D)
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