Secondary antibody inucubation The horseradish peroxidase (HRP)-labeled secondary antibody is diluted with a blocking solution in an appropriate ratio with reference to the instructions for the secondary antibody. After draining the washing solution, immediately add the diluted secondary antibody, incubate for 1 to 2 hours at room temperature or 4 ° C on a shaker with gentle shaking. Recover the secondary antibody. The Western wash is then added and shaken slowly on a shaker for 5-10 minutes. After draining the washing solution, the washing solution is added and washed for 5-10 minutes. Washed a total of 3 times. If the result background is higher, the washing time can be appropriately extended and the number of washings can be increased. The first 2 steps of washing are to wash away the non-specific binding of the primary antibody and the secondary antibody. The washing effect directly affects the depth of the background of the result, so the washing must be clean. The wash solution uses TBST or PBST, where Tween20 helps remove non-specific binding and reduces background. At the same time, washing the film several times in a short time (such as 5-6 times, 3 minutes each time) is more effective than washing the film a few times for a long time. The third step of TBS washing is to wash away the Tween-20 on the membrane because it can hinder the deposition of the substrate. Precautions 1) Precautions for antibody storage: 2) After receiving the antibody, centrifuge as required and then open the tube cover for dispensing and storage! 3) For most antibodies, a suitable storage method is to store at -20 °C after dispensing. 4) The amount of dispensing should be used up in one experiment, at least not less than 10 ul 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. 5) If the reconstituted antibody is not used at one time, keep the remaining mother solution at 40 °C to avoid freezing again! 6) Never avoid storing antibodies in an automatic defrost refrigerator. 7) The transient storage of most antibodies at 40 ° C for 1-2 weeks has no effect on antibody activity. 8) Long-term storage, adding azide to prevent bacterial contamination. Primary/secondary use problem: The concentration of the primary antibody and the secondary antibody is generally selected according to the antibody specification, and the selection of the primary antibody directly affects the experimental results and the depth of the background. To strictly ensure the reaction time, the membrane should be washed as much as possible to wash the primary antibody, which is beneficial to reduce the background; also pay attention to the matching of the primary antibody. Natural and non-reduced sample problems: Some antibodies only recognize epitopes that are non-contiguous amino acids. Although these amino acids are not continuous with each other in the primary structure of the protein, they are close together in the spatial structure. These antibodies only recognize the spatial structure of the target protein (specified in the antibody specification). For these samples, the buffer and gel should not contain SDS and could not heat denature the protein. Some antibodies only recognize the non-reducing state of the protein, such as the oxidized state. For such samples, it is necessary to remove the reducing agents DTT and beta-mercaptoethanol in the buffer and gel. For more information, please visit Leather Fashion Gloves,Half Gloves Fashion,Stylish Leather Gloves,Hand Gloves For Fashion HOLIK ASIA GROUP CO.LTD , https://www.holikasia.com