Mouse IL-17 ELISA Kit (ab100702)

When reconstituting your standard, we suggest that after it has been thoroughly defrosted, spin the vial down, reconstitute with assay diluent and then spin down again before use.
When making your standard dilutions, we recommend keeping everything on ice because diluted standards are not stable and need to be used on the same day. When preparing these dilutions mix the standard thoroughly with your pipette by pipetting up and down but do not vortex the solution as this may damage the protein.

Thank you for contacting us.
This ELISA kits is specific to IL17A. It does not recognize IL-17B, C, D or F.
Please let me know if you have any questions or comments.

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He querido comprobar la posibilidad de presentar reactividad cruzada entre ratón y humano en los kits contra las citoquinas que mencionas.

Tenemos varios kits contra dichas citoquinas específicos para ratón, y específicos para humano. Después de estudiar la homología entre secuencias, me temo que no podemos predecir que exista reactividad cruzada entre especies. Experimentalmente tampoco tenemos datos para confirmarlo.

Siento no poder ser de mas ayuda. No dudes en contactarnos para cualquier otra consulta.

Thank you for your inquiry.

Unfortunately, for the antibodies in IL17 Mouse ELISA Kit ab100702 epitope mapping has not been performed, so we do not know the exact region the antibodies are binding to the mouse IL-17A protein. However, I can let you know that the antibody pair is binding between amino acids 26 and 158 of mature mouse IL-17A as the whole mature protein used as immunogen.

We aim to provide as much information as possible to customers, so I am sorry that this has not been possible on this occasion. I hope this information is nevertheless helpful to you. Please do not hesitate to contact me if you have any further questions in this regard.

Following up my previous message, here are some recommendations for preparing cell and tissue lysates for use with this kit, though they are not specific for oviducts or lymph nodes: 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 strongly recommend that you add some type of protein 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 protein 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 (using a Bradford-Lowry-type 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 1x106 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. We recommend diluting the resulting samples at least 5-fold with Assay Diluent B to minimize any effects of the detergents in the lysis buffer. The samples may need to be diluted further but this would need to be determined empirically. So for the original lysates, you want to aim for at least 1 mg/ml protein, preferably more, so you can achieve ~50-500 ug/ml after dilution. I hope this is helpful. Please let me know if you have any other questions.

Thank you for contacting us. Can you please reply with a few details of the trouble you are having with the standard curve? For a positive control, I suggest isolating lymph nodes from a mouse, if possible. For the oviduct homogenate preparation from frozen tissue (and also the lymph node preparation), simply thawing the samples on ice in a small volume of cold buffer with protease inhibitors may be sufficient to lyse the cells in the tissue and release the IL17. However, you may want to include some detergent, for instance 0.1% Triton X-100. Follow with sonication for several seconds and then spin down to remove insoluble material. I am going to ask the lab for more specific suggestions and I will forward these when I receive them.