When we want to detect the expression of the protein of interest in the tissue, how to choose the antibodies that are suitable for our experimental needs, and properly preserve the use, every researcher needs to carefully consider. There are many kinds of antibodies, many brands, different technical parameters, and the price and quality are very different. How to buy high-quality and suitable antibodies and accurately make the results we want, there are many details to be aware of.
First, how to choose the antibodies that are suitable for your experiment?
1. Primary resistance selection points
(1) Determine the name of the antibody, pay attention to the Chinese and English names, its name, subtype and other information.
(2) Determine your type of experiment, Elisa, WB, IHC, ICC, or FACS. The general antibody instructions will list the type of antibody the antibody has been validated for. If the type of application not mentioned in the antibody specification does not imply that the antibody is not suitable for this type of analytical application, it is only After verification of this type of experiment, please select the antibody that is suitable for your experiment according to the type of verification listed in the manual.
(3) Determine the species of the experimental sample, Human, Mouse, or Rat. The general antibody instructions list which species of the antibody the test has been tested for. Please select the validated species according to the instructions to select the appropriate antibody for your experiment.
(4) Structural properties of the sample protein. Understanding the structural properties of the sample protein helps to select the most suitable antibody. Whether the domain and sample of the sample protein to be tested will be denatured during extraction and processing, and the change of the protein space conformation will affect the immunoaffinity reaction of the antibody.
(5) Selection of single polyclonal antibodies. The antibodies on the market are mainly polyclonal antibodies. Generally, the monoclonal antibodies are specific, but the affinity is relatively small, and the sensitivity of the detection antigen is relatively low. The specificity of the polyclonal antibodies is slightly weak, but the affinity of the antibodies is strong and the sensitivity is high. However, non-specific staining is easy to occur (can be avoided by blocking, etc.).
2. Secondary resistance selection points
(1) Source of species. The source of the secondary antibody is determined mainly according to the source of the primary antibody. For example, the primary antibody is the source of the mouse, and the secondary antibody can be purchased from the anti-mouse (the sheep, the rabbit, etc.).
(2) Selection of markers. There are markers such as HRP, Biotin, and fluorescein. Generally, the SP three-step secondary antibody selects the Biotin-labeled secondary antibody to bind to the latter SP, and immunofluorescence staining requires the purchase of different fluorescein-labeled secondary antibodies, such as rhodamine, FITC, Cy3 and the like.
Second, the stability of the antibody
1. The stability of antibodies is the result of long-term evolution in nature. Antibodies are one of the most important molecules in the immune system, and their stability is the result of natural evolutionary screening. In many species, antibody molecules form a conserved and stable structure.
2. High Tm values ​​of antibody molecules. At room temperature, even at short-term temperatures of 50-60 ° C, antibody molecules can maintain proper folding and maintain the desired activity.
3. The purity of the antibody. Advanced antibody purification technology ensures high purity of antibody products and ensures the stability of their antibody products.
4. The concentration of the antibody. The high concentration of the antibody product can reduce the material loss caused by the adsorption on the surface of the container, and the high concentration of the antibody is more stable, and the concentration of 93% in the antibody product is greater than 0.5 mg/ml.
Third, how to determine the antibody purchase channel and brand?
The quality of famous foreign antibody brands is generally no problem, but the price is more expensive, and many antibody brands do not produce antibodies themselves, so we can find imported antibodies produced by OEM antibody manufacturers. With the increasing popularity of network technology, we ourselves You can search through the relevant websites at home and abroad to find the product information we need.
Fourth, how to save the obtained antibodies?
Whether the antibody is preserved or transported, it should avoid freezing and thawing as much as possible. After receiving the antibody, please separate the antibody according to the needs of the experiment to avoid repeated freezing and thawing of the same antibody (repeated freezing and thawing will cause the spatial structure of the antibody to be denatured. Lead to the formation of multimers, thereby reducing the binding capacity of the antibody). Whether the antibody is stored properly or not directly determines the activity and use of the antibody. If the antibody is properly stored, most of the antibody activity can be maintained for several years. Please keep in mind that whenever you follow the recommended storage conditions, the antibody should be handled and stored correctly.
1. After receiving the antibody, be sure to centrifuge at 4 ° C, 12000 rpm for 3 minutes (whether liquid or lyophilized powder), then open the tube cover for dispensing and storage (if the antibody volume is less than 50 ul, please extend the centrifugation time) Up to 5 minutes to ensure that all antibodies are centrifuged). The antibody is usually made of a special storage tube. Only after centrifugation at 12000 rpm for 3 minutes can all the antibodies adhering to the tube wall or the lid be centrifuged (even if the centrifugation is not complete at 10,000 rpm, resulting in insufficient antibody). .
2. For ProSci most antibodies, the appropriate storage method is to store at -20 ° C or -80 ° C after dispensing.
(1) For most antibodies, storage at -20 °C is sufficient, and there is no evidence that it is better to store at -80 °C.
(2) Packing can minimize the damage of antibody activity caused by repeated freezing and thawing, and also reduce the possibility of contamination caused by multiple times of taking antibodies from the same tube.
(3) The amount of dispensing should be used up in one experiment, at least not less than 10ul per serving. Because the smaller the volume of the package, the more likely the concentration of the antibody is to be affected by evaporation and wall adsorption.
(4) If the dispensed antibody after reconstitution is not used at one time, store the remaining mother solution at 4 °C to avoid further freezing.
(5) The antibody working solution should be used up on the same day of the day, and should not be stored for more than 1 day at 4 °C.
(6) Absolutely avoid storing the antibody in the automatic defrost refrigerator, and keep the antibody in the inner layer of the refrigerator to avoid repeated freezing and thawing caused by temperature changes.
3. Most of the antibodies were stored at 4 ° C for 1 to 2 weeks after receiving, and had no effect on antibody activity.
4. Transportation conditions of most antibodies: The general transportation process takes 1~2 weeks, so it is completed under the condition of low temperature 4 °C.
The main purpose of 4°C transportation is to avoid the damage of antibody activity caused by repeated freezing and thawing. (If using dry ice transport, the antibody is frozen when received, and needs to be thawed for dispensing. This is an additional freeze-thaw process. ), so dry ice transportation is absolutely avoided during transportation.
About the use of sodium azide:
In order to prevent microbial contamination, sodium azide is often added to the antibody (final concentration 0.02% (w/v)). The vast majority of antibodies to ProSci already contain 0.02% to 0.05% sodium azide, as indicated in the storage buffer of the instructions.
Note: How to remove sodium azide
It can be removed by gel electrophoresis or filtration. The molecular weight of IgG is 150kDa (IgM is about 600kDa); the molecular weight of sodium azide is only 65Da. The sodium azide can be removed from the antibody by using a filter with a molecular weight cut off of 14kDa. .
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