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I am interested in measuring mouse IL-1 beta in samples of plasma and tissue, is it possible to use only one type of kit (ab100704 or ab100705) for both? Which is the difference between them?
Asked on Sep 09 2015
It is possible to use the non-lysate kit ab100704 (IL-1 beta (Interleukin-1 beta) Mouse ELISA Kit) and use your own lysis buffers (non-denaturing, with a mild detergent like RIPA/NP40/Triton X). The cell lysate kit (ab100705) contains this buffer and a different assay diluent, but the different assay diluent is not required to run the assay correctly with tissue/cell lysate samples.
I can recommend to use a detergent based on our sample preparation tips here:
Avoid using >0.1% 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.
Use no more than 2% v/v total detergent
Avoid the use of sodium azide
Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols
We strongly recommend adding a 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 forms of the protein.
Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no best method for all 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, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10 min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any immunoassay. Next, determine the protein concentration of your lysates using a total protein assay not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay. Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.
Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 µL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target total protein concentration of the homogenate should be at least 1,000 µg/mL, but 2,000 µg/mL or more would be better.
Anja HoffmannAbcam Scientific Support
Answered on Sep 09 2015