There are always big or small problems during the operation of the Elisa kit, such as flower plates, false positives, full color rendering, lower signal values ​​than blanks, and so on. Today, Shanghai Hengyuan Bio brings us some experimental experience, and the Elisa kit experiment will not fail:
1. Some of the original coatings may not be proteins. For biotin and lipid substances or small molecular substances, we have to modify and then apply them. I will list the methods as follows:
2. Avidin biotin: The first avidin is coated with a carrier and biotinylated DNA is added. This coating method is average and firm, and has been expanded to be used for quantitative determination of various antigenic substances.
3. Small molecules must be coupled to a large protein carrier to be immobilized on a solid support. The nature of the original coating is very important, the protein concentration, whether it is degraded, which is related to whether the antibody you made can be recognized by it, so it is very important to retain the antigen. When I am doing recombinant protein, my brothers strictly warn me that I must be in the ice bath. Slow melting is the reason.
4. Lipid substance: It can be dissolved in an organic solvent, added to the hole of Elisa plate, opened and placed in a refrigerator overnight or blown dry by cold air. After the alcohol is volatilized, the lipid is naturally dried on the surface of the solid phase.
5. The following principle should be noted: because the protein and polystyrene solid phase carrier are bound by physical adsorption, relying on the interaction between the hydrophobic group on the structure of the protein molecule and the hydrophobic group on the surface of the solid support, this physics The adsorption length is specific, affected by the molecular weight, isoelectric point, concentration, etc. of the protein. Large molecular proteins usually contain more hydrophobic groups, so they are more easily adsorbed onto the surface of the solid support. For the choice of coating solution, ph9.6 carbonate buffer is generally selected. However, sometimes due to the needs of the test, the specificity of the original coating may also be packaged with a neutral buffer solution. Detailed tests must be practiced in addition to solid theory. See if the ultimate can be applied to your own experiments. Commonly used coating solutions include pH 7.2 phosphate buffer and pH 7-8 Tris-HCL buffer, etc., in addition to the pH 9.6 carbonate buffer just mentioned.
6. But what is the ultimate choice, should be based on the test details. Commonly used blocking agents are: 0.05%-0.5% BSA; 10% calf serum or 1% gelatin; skim milk powder, relatively inexpensive, can be used at high concentration (5%-10%); there are some rare useful Various animal serums (mainly to rule out similar protein interference) and casein and the like. Blockade is the process of filling a large amount of unrelated protein with these sputum vacancies, thereby rejecting the re-adsorption of interfering substances in the steps following the ELISA kit. Blockade: The process of recoating with a high concentration of unrelated protein solution after coating.
7. There will be some errors in the washing of the board, the human factor is very large (except for the premise of using the washing machine), the washing is not complete or stringed holes, it is not a small impact on the agile ELISA system. Since the adsorption of proteins by plastics such as polystyrene is universal, in order to achieve separation of free and bound enzyme labels, free substances remaining in the pores of the plate, as well as non-specifically adsorbed interfering substances, are washed. This non-specifically adsorbed interfering substance should be washed away. Washing plate: It can be said that washing is the most important pivotal technique in ELISA manipulation.
8. Add antibody specimens (and secondary antibodies): Note that the tip of the gun should be changed when changing the tip. Specimen dilution can be diluted with pbs or diluted with blocking solution. If you need to add a secondary antibody, you should also pay attention to the working concentration of the secondary antibody, which is too high and waste. If it is too low, the coloration is shallow.
9. Color development: There are many color development systems. When we start, we should choose the appropriate color development system. Note the preservation of the enzyme-active substrate of the chromogenic system. The HRP conjugate plus the sulphuric willow pump, the AP conjugate can be added with sodium azide.
10. Do as much as possible to do the three comparisons of yin and yang, and analyze the problem if there is a problem.
More Elisa technology sharing, all in Shanghai Hengyuan Biotechnology Co., Ltd.!
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