Mouse ICAM1 ELISA Kit (CD54) (ab100688)

Thank you for contacting us. Althought this particular kit has not, to our knowledge, been tested with tissue homogenates, it should be compatible.

We recommend diluting the samples at least 5-fold with Assay Diluent D 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 by the experimenter empirically.

In brief, a lysis buffer must meet the following specifications:

A) has relatively low salt content (700 mM or less)

B) does not contain sodium azide

C) does not contain >0.1% SDS

D) does not contain >10 mM reducing agents (beta-mercaptoethanol or dithiothreitol)

For the original lysate, in general you want to obtain a stock concentration at least 1 mg/ml protein, preferably more, to achieve 50-500 ug/ml after dilution. This range should just be used as a starting point and the optimal concentration would need to be determined empirically.

Here are more general instructions from the laboratory that developed this kit.

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 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 1e6 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.

Thank you for contacting us.

The following ELISA kits for mouse ICAM, VCAM, IL-6 and TNF-alpha are suitable for mouse samples:

ab100688 (https://www.abcam.com/ICAM1-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100688.html)
ab100713 (https://www.abcam.com/IL6-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100713.html) was tested on tissue as well.
ab100747 (https://www.abcam.com/TNF-alpha-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100747.html)
ab100750 (https://www.abcam.com/VCAM1-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100750.html)

Please let me know if these are kits you were refering to.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for your call earlier this week and for your patience while I have looked into your enquiry. I contacted the lab to ask about cross-reactivity of our mouse ICAM1 and VCAM ELISA kits with the other CAM's, and unfortunately the antibodies in these kits are not expected to cross-react with VCAM, ICAM, and PCAM. I am sorry that we do not have the product you are looking for, but if you have any questions or need anything further please let me know.