小鼠TIMP1 ELISA试剂盒(ab100745)

Thank you for contacting us. I would suggest using ab100745 TIMP1 Mouse ELISA Kit - 1 x 96 Well Plate. This has been specifically designed for lysate and tissue samples, and optimized to reduce background that can sometimes occur. I have attached some general tips to help prepare the lysate samples. Please note that this is just a general guideline and the optimal method should be determined based on researched literature and knowledge of the target and samples. Finally, to minimize any effects of detergents in the prepared lysate samples, we advise diluting them at least 5X with the appropriate diluent but further dilution may be needed. Please let me know if you have any questions or comments. How do I make cell or tissue lysates for use on the cytokine arrays? 1) Avoid using SDS or other strongly denaturing detergents. In general, non-ionic detergents, such as Triton X-100 or NP-40 are best, although zwitterionic detergents, such as CHAPS, or mild ionic detergents, such as sodium deoxycholate will work. 2) Use no more than 2% v/v total detergent 3) Avoid the use of sodium azide 4) Avoid reducing agents, such as dithiothreitol or mercaptoethanols In general, we recommend that you add some type of protease inhibitor “cocktail” to the lysis buffer prior to homogenization. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used, but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated (activated) forms of the protein. Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freeze-thaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no one “best method” for all sample types, but some are better than others for some sample types. Your choice of method should be made following a brief search of the literature to see how samples similar to yours have been prepared in previous investigations. After homogenization, spin down the lysates to remove cell/tissue debris (5 min @ 10000 x g or 10 min @ 5000 x g) and save the supernatant. Lysates should be frozen as soon as possible, and stored at -20°C (or -80°C, if possible). Spin them again before incubating with the antibody array. Determine the protein concentration of your lysates (for example the bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay. Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 uL of lysis buffer per 1x10^6 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target for total protein concentration of the homogenate should be at least 1000 ug/mL, but 2000 ug/mL would be better.