Loading control western blot step12/18/2023 ![]() Radioimmunoprecipitation (RIPA) buffer and NP-40 buffer are common lysis buffers and mostly differ based on the type of detergents used for extraction and solubilization of proteins. ![]() Ideally, lysis buffer should be robust enough to extract a sufficient amount of protein, yet provide conditions that retain the native structure of the protein of interest and not disrupt specific protein-protein interactions. ![]() Various factors influence the choice of lysis buffer, including the location of the protein of interest (i.e., cytosolic, membrane-bound, or within subcellular structures such as the nucleus or mitochondria), solubility of the protein in different detergent types, and compatibility of the buffer in the downstream IP application. Additional physical methods can be used in combination with reagent-based lysis methods when desired or necessary. If preparing your own cocktails, follow the manufacturer’s instructions and recommendations for stock solutions and working concentrations of your specific protease targets, as the half-life of different inhibitors can vary. For convenience, many manufacturers offer easy-to-use pre-prepared cocktails. Lysis buffers must contain fresh protease and phosphatase inhibitors (such as in cocktail form), as degradation and dephosphorylation can begin immediately upon lysis. Prepare cell or tissue lysates using standard methods and be sure all lysate collection steps are performed on ice or at 4☌. Tips for Immunoprecipitation Using TrueBlot® Products Skip to: Tips for Western Blotting using TrueBlot® Products ![]() Check out the tips below to get the best results when using TrueBlot® products in IP or Western blot assays. When paired with TrueBlot® IP beads, the TrueBlot® product line allows you to generate reproducible, publication-quality IP/Co-IP-Western blots. The unique detection properties of TrueBlot® secondary antibodies recognize the native (non-reduced) form of IgG over the reduced form of IgG to eliminate interference from the denatured heavy and light chains of the IP antibody. Rockland’s TrueBlot® product line is designed to solve the common experimental problems encountered when performing Western blots of Immunoprecipitated (IP) or Co-Immunoprecipitated (Co-IP) samples. ![]()
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