#elisa plate

[lovedria-blog]

Why is the ELISA plate blocked in the elisa experiment?

In order to make the results more accurate. If it is not closed, there may be an oxidation reaction with oxygen in the air, or other reactions, or pollution, which will cause deviations in the results.

[ivymaid]

Is there any difference between a 96-well microplate and a 96-well plate for cell culture?

The bottom of the ELISA plate of different ELISA kits is different, and there are processed ones inside. The bottom of the cell culture plate is empty. Transfer the liquid to the microplate reader for testing. It is just a container.

[lubeytunes]

Can the microtiter plate be reused?

If your enzyme labeling reaction has entered the final step and added the stop solution 2M sulfuric acid, then I suggest not to reuse it. The stop solution will stop the reaction and invalidate your coated antibody or antigen.

If you want to reuse it, you can only use a polystyrene microtiter plate with no specific solid support, which can be obtained by removing the coating, and then re-coating the target antibody or antigen for reuse. This process is not practical for routine testing. Significance, and the low price of solid-phase carrier polystyrene microtiter plates, not most people use.

[shelbysdreams]

What is the difference between microplate and multi-well cell culture plate?

The microplate reader can measure the absorbance, but only microplate and 96-well cell culture plates. The microplate is mainly used for ELISA. The cell culture plate is mainly used for culturing cells and then adding MTT, CCK8, etc. to make living cell mitochondria A ginseng is produced and colored. In addition to 96-well multi-well cell plates, there are 6, 12, and 24 wells.

[heyleafmealone]

Classification of cell culture plates and the difference between them and microplates

The cell culture plate, as the name suggests, is used to cultivate cells. According to the culture method, it can be divided into suspension culture and adherent culture (the surface is processed for adherence culture). According to the bottom shape, it can be divided into flat bottom, round bottom, and V-shaped bottom. Generally, adherent culture is used Flat-bottomed culture plates, V-bottom and U-bottom are generally used for suspension culture. According to the number of holes, it can be divided into 6 holes, 12 holes, 24 holes, 48 ​​holes and 96 holes. The specific selection depends on the type of cultured cells, the required culture volume, and different experimental purposes.

The different bottom culture plates have their own characteristics, and their applications are also different. Generally, the flat-bottomed culture plate we use (some manufacturers also call it F-type bottom) is mainly used for flat-bottomed assays, such as cell culture; U-shaped bottom culture plates can provide the best cleaning effect and mixing performance, and are suitable for testing. Application of sediments; V-bottomed culture plates are suitable for sedimentation applications or applications for optimal small-volume recovery.

The material of the cell culture plate is generally polystyrene, which is the same as the material of the microtiter plate, but it is essentially different from the microplate. Cell culture plates are mainly used for cell culture and can also be used to measure protein concentration; while enzyme-labeled plates are used for enzyme-linked immunoassays, which have higher requirements and are generally more expensive than cell culture plates.

[loirafey]

What is an ELISA plate?

It is a kind of equipment used for enzyme-linked immunoassay in conjunction with the microplate reader. Although it looks like an ordinary plastic plate with holes, it is an indispensable part in enzyme immunoassay.

Enzyme-linked immunosorbent assay, referred to as ELISA, is the most widely used technique in enzyme immunoassay technology. The basic method is to adsorb a known antigen or antibody on the surface of a solid carrier and maintain its immunological activity. Then the antigen or antibody is linked with a certain enzyme to form an enzyme-labeled antigen or antibody. This enzyme-labeled antigen or antibody retains its immune activity and enzyme activity. In the measurement, the test specimen (the antibody or antigen in the measurement) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid-phase carrier in different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing, and the amount of enzyme bound on the solid-phase carrier is finally proportional to the amount of the test substance in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the test substance in the sample, so it can be qualitatively or quantitatively analyzed according to the shade of the color reaction.

You can see that there is a "solid phase carrier" in this process, which is the microplate. So it can be known that the microtiter plate is an indispensable intermediary. Without this intermediary, the entire experiment cannot be operated. In other words, as long as you use a microplate reader to do an enzyme-linked immunoassay, you will inevitably use the microplate.

[millathememe]

Summary of ELISA experience:

1. The nature of the coating source is very important. The protein concentration and whether it is degraded are related to whether the antibody you make can be recognized by it. Therefore, it is very important to preserve the antigen. When I make recombinant protein, my seniors strictly warn me to This is why it slowly melts under the ice bath. There are also some coating sources that may not be proteins. For biotin and lipids or small molecules, we need to modify them before coating. I briefly list the methods as follows:

①Avidin Biotin: Pro-avidin first coats the carrier and adds biotinylated DNA. This coating method is uniform and firm, and has been expanded to be used in the quantitative determination of various antigen substances.

② Lipid substances: Dissolve them in organic solvents (such as ethanol) and add them to the wells of the ELISA plate, open the lid and leave in the refrigerator overnight or dry in cold air. After the alcohol evaporates, let the lipids dry naturally and solidify on the solid surface.

③Small molecules must be fixed on the solid-phase carrier only after being coupled with the large protein carrier.

2. The choice of coating liquid, generally choose ph9.6 carbonate buffer. But sometimes due to the needs of the experiment, the particularity of the coating material may also be coated with a neutral buffer solution. The following principles should be noted: since the protein and polystyrene solid-phase carrier are combined by physical adsorption, it relies on the interaction between the hydrophobic groups on the protein molecular structure and the hydrophobic groups on the surface of the solid-phase carrier. This physical adsorption is Non-specifically, it is affected by the molecular weight, isoelectric point, concentration, etc. of the protein. Smaller macromolecular proteins usually contain more hydrophobic groups, so they are easier to adsorb to the surface of the solid support. Specific experiments must have a solid theory and practice, and see if they can be applied to their own experiments in the end. In addition to the pH 9.6 carbonate buffer just mentioned, the commonly used coating solutions also include pH 7.2 phosphate buffer and pH 7-8 Tris-HCL buffer.

[inlove-withnyc06]

Is the protein easier to bind to the microplate when the pH is alkaline?

The material of the ELISA plate is mostly polystyrene (PS). The binding of PS and protein is through physical adsorption. It relies on the hydrophobic groups on the protein molecular structure and the hydrophobic groups on the surface of the solid carrier. Inter-group force. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, and concentration of the protein.