For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
LOT NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I would like to know if this ELISA (ab100705) is specific for IL-1 beta the mature form or if it can cross-react with pro-IL-1 beta or IL-1 alpha. I know there are both present and wonder if this is causing issues. Also, do you have a protocol/suggestion of how to lyse mouse melanoma (tissue) samples prior to ELISA analysis? do you know of any publications that have used this assay to quantify IL-1beta in mouse tissues? thank you! SAMPLE mouse melanoma B16F1 tissue sample PRIMARY ANTIBODY as in protocol DETECTION METHOD as in protocol POSITIVE AND NEGATIVE CONTROLS USED which would you suggest? negative obviously no sample added and biological control tumour without treatment ANTIBODY STORAGE CONDITIONS as recommended TYPE OF ELISA it's your elisa - you know COATING WELL as in protocol BLOCKING CONDITIONS as in protocol SECONDARY ANTIBODY as in protocol
Asked on Feb 06 2012
Thank you for your email.
I can confirm that this kit will recognize the mature form (aa 118-269) of IL1beat.
Since the pro IL1beta also contains this amino acid sequence, I would expect the kit to also detect the pro-form. This has not been experimentally tested by us.
The IL1 beta Mouse ELISA Kit does not cross-react with IL1alpha. This has been experimentally tested by the lab.
I have to confirm that ab100705 has not yet been referenced specifically in any publications we know of.
I copied our suggestions for the preparation of tissue lysates below.
I hope this information is helpful. Please do not hesitate to contact me again with any further questions.
How do I make cell or tissue lysates for use on the cytokine arrays?
The cell or tissue lysates for use with ab100705 ELISA kit can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply a compatible lysis buffer in most of our kits. Other general low-salt lysis buffers can be used with the following caveats:
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.
Answered on Feb 06 2012