The principle of the ELISA experiment seems very simple, nothing more than fix the antigen, add the primary antibody, secondary antibody and substrate, intermittently interspersed with washing and blocking. However, even the plain washing and closing, if not done well, may ruin the whole experiment. At the end of the experiment, whether we can obtain meaningful information depends largely on the signal-to-noise ratio of the results. Background noise can affect your judgment of the results. Here are some tips on how to reduce the background of ELISA. Washing is important The washing step may seem boring, but it is important because if unbound material (such as non-specifically bound antibodies or detection reagents) remains in the microplate, it will increase background noise. If necessary, increase the salt concentration in the wash solution, which will prevent non-specific binding reactions. If the background is too high, and you suspect that the washing is not enough, you can try to increase the number of washing. Closed is more critical The role of the blocking solution is to allow unrelated proteins to occupy potential binding sites in the microplate. This reduces the chance of non-specific binding of antibodies that can generate signals. Of course, you also hope that the antibody only binds to the target protein. If your background is too high and you suspect that the blocking is not sufficient, then you can try to use a higher concentration of blocking solution or extend the blocking time appropriately. If you have been troubled by background problems, then maybe you should spend some time optimizing the blocking solution. This may take time, but it is also worth it. There are currently two main types of blocking solutions: protein and non-ionic detergents. The type you use depends on many factors, including the surface reagents of the microplate, the adsorbed antigens, your antibodies, and detection reagents. A good blocking solution should reduce non-specific binding, but it should not interact with antigens, antibodies, or detection reagents. The most commonly used non-ionic detergent is Tween-20. This blocking solution is cheap, stable, and useful for removing some non-specific binding during the washing process. But they only work when they exist, because they are easily washed away. Therefore, blocking solution must also be added to all washing solutions. Do not use high concentration (normal concentration is 0.01-0.1%), it will reduce specific binding and produce false negatives. Another option is to use two blocking solutions, protein and non-ionic detergents, the latter assisting in blocking during washing. The protein blocking solution is different and permanent. They bind to open sites and block, while stabilizing antigen molecules bound to microplates. Commonly used protein blocking solutions include bovine serum albumin (BSA), skimmed milk powder, normal serum and fish gelatin. The inherent diversity of serum components allows it to effectively intercept many different types of molecular interactions. However, its disadvantage is that it can interact with Protein A and IgG antibodies. One way to solve this problem is to use normal serum from chicken or fish. Antibody concentration must be optimized We usually follow the steps left by Brother and Sister, but if the reagents are slightly different, you may need to optimize the amount of antibody. Remember, non-specific antibody binding increases background. To prevent this, never use too much primary or secondary antibodies. Appropriate amount of detection reagent Another point is also obvious: do not use too much detection reagents. If the concentration is too high, or if it is not properly diluted, it will result in high background. Do not over-develop the color, if necessary, optimize when the stop solution should be added. If your ELISA unfortunately encounters a high background, then do n’t worry too much. Look at the components in the ELISA system in turn and eliminate possible problems. Optimize carefully, I believe there will be beautiful results soon. Hair Curved Scissors,Baber Scissors Set,Customized Professional Scissors,Custom Salon Hair Scissors Zhangjiagang Mister Tools Co., Ltd , https://www.mingshitools.com