If desired results are not obtained, test several different dilutions of the primary antibody, or try a different dilution buffer. When using an antibody for the first time, choose a concentration based on the literature or stated on the product datasheet. Determining an optimal concentration of the primary antibody If it’s too high, non-specific reactions occur, resulting in detection of non-target proteins. If it’s too low, the target protein cannot be detected. The concentration of the primary antibody is critical. ※PBS-T (PBS-Tween 20): Phosphate-buffered saline (PBS) containing 0.05% Tween 20 Probing with antibodiesįollowing blocking, the membrane is probed with a primary antibody, and then with a secondary antibody. Skim milk contains the phosphoprotein casein, and therefore is not suitable for the detection of phosphoproteins. However, it can interfere with specific antigen-antibody reactions. Skim milk is more effective in blocking and used frequently. Blocking and probing with antibodies Blockingīovine serum albumin (BSA) is used as a blocking agent. When transferring high molecular weight proteins, reduce the methanol concentration to allow the gel to swell slightly, which facilitates protein transfer. Methanol stabilizes the gel and prevents gel from swelling. Composition of transfer buffer (Commonly used) The composition of a commonly used transfer buffer is shown below. Transfer bufferĭifferent buffers are used for transfer, depending on the molecular weight and the nature of the target proteins. ※Re-probing: Stripping the membrane of antibodies after detection, and probing with another antibody. Nitrocellulose membrane is more fragile but less expensive than PVDF, and does not require pre-wetting with methanol or cause as much non-specific binding. Though more expensive, the PVDF membrane is stronger and has a high absorption capacity with proteins, which makes it suitable for CBB staining, detection with certain substrates for alkaline phosphatase, and re-probing. Nitrocellulose and PVDF membranes are commonly used. Comparison between blotting apparatuses Membrane A semi-dry or a tank system can be used for transfer. Proteins separated by SDS-PAGE are “transferred” from the polyacrylamide gel to a membrane, using a specialized apparatus (blotting apparatus).
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