![]() ![]() Standard immunodetection, however, offers higher sensitivity and requires less optimization for new sample types. The rapid immunodetection method works well to quickly visualize higher abundance proteins. Rapid immunodetection eliminates the blocking step and reduces the time necessary for the washing and incubation steps. Incubating the membrane with a substrate that reacts with the conjugated secondary antibody to reveal the location of the protein.Washing to remove any unbound secondary antibody.Incubating the membrane with a conjugated secondary antibody, which binds the first antibody.Washing to remove any unbound primary antibody.Incubating the membrane with primary antibody, which binds to the protein of interest.Blocking unoccupied membrane sites to prevent nonspecific binding of antibodies.Standard Immunodetection Methods Include the Following Steps: There are two types of protocols for immunodetection: Standard and rapid. However, to achieve the highest specific signal and lowest non-specific background signal, the best antibody concentration, incubation temperature, and incubation time must be determined individually.New protocol saves time and delivers consistency. Note: The SYSY standard protocol generates good results in the SYSY labs and may be used as a reference. Stop staining reaction by washing 3 times with H 2O.Time can be shortened or extended if signals are extremely strong or weak, resp. Replace with fresh staining solution complete and develop for 15-30 min.Wash with substrate buffer and equilibrate for 5 min.Wash 3 times with 5% skimmed milk-TBST for 10 min each time.Incubate with fresh 5% skimmed milk-TBST containing the recommended AP-conjugated secondary antibody (anti-mouse IgG, anti-rabbit IgG, resp.) at the appropriate dilution for at least 1 h on a lab shaker.Wash 3-4 times with 5% skimmed milk-TBST for 10 min each time.Incubate in fresh 5% skimmed milk-TBST containing the primary antibody at the appropriate dilution for at least 2 h on a lab shaker at RT or over-night at 4☌.Rinse the membrane in water to remove the Ponceau S staining solution and incubate in 5% skimmed milk-TBST for 30 min on a lab shaker at RT.Stain the membrane with Ponceau S staining solution for several minutes at room temperature to check the efficiency of transfer.Follow the manufacturer's instructions for your SDS-PAGE and blotting device. Separate the protein sample to be examined and a molecular weight standard using SDS-PAGE and transfer to a nitrocellulose membrane by electro-blotting. Alkaline phosphatase (AP) conjugated secondary detection reagent.Prepare this solution shortly before use. Staining solution complete: Substrate buffer containing 80 µl BCIP solution and 60 µl NBT solution per 10 ml.NBT staining solution: 50 mg/ml in 70% di-methyl formamide.BCIP staining solution: 20 mg/ml in 100% di-methyl formamide.Substrate buffer for alkaline phosphatase: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2.5% skimmed milk in Tris buffered saline with Tween 20 (5% skimmed milk-TBST): 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skimmed milk powder, 0.02% sodium azide, 0.1% Tween 20.Ponceau S staining solution: 5% acetic acid, 0.1% Ponceau S.Please refer to the remarks sections for western blotting on the respective data sheet. unboiled samples or special gel systems). Important: Some proteins have special requirements for good separation (e.g. Compared to enhanced chemiluminescent (ECL) detection, AP staining is less sensitive, but does not require special imaging equipment for visualization of the assay results. The color precipitate can easily be observed during development, and the staining reaction can be stopped when the desired signal strength is reached. In standard Western blot (WB) approaches, protein samples are separated according to their molecular weight with denaturing SDS-PAGE (polyacrylamide gel electrophoresis), transferred to a membrane and analyzed by immuno-detection with antibodies.Ĭhromogenic alkaline phosphatase (AP) staining is a cumulative detection system. ![]()
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