I am studying the pathogenesis of bacterial toxins in my laboratory. In particular, I am interested in the cytokine responses elicited in endothelial cells after treatment with a specific toxin. I did not want to do the western blots or ELISAs one after the other and had a desire to look at a complete cytokine profile. While looking around for the right product, I came across the cytokine antibody array by RayBiotech. Since proteins execute all cell functions, it is more beneficial to look at the protein level than at the mRNA level. Raybiotech has, for the first time, come up with a protein array that is convenient to use. It does not require any special equipment and if a lab has the basic facilities to do western blots one can handle these arrays as well. Depending on your needs you can select either human or mouse cytokine arrays. Arrays can be used with conditioned medium, serum, plasma or cell lysates. I used the Human cytokine antibody array 3.1 where I planned to use cell lysates. The detection range is about 100 fold greater than ELISA and you can also get a custom array made suitable to your requirements.
The kit includes cytokine array membranes, biotin conjugated anti-cytokines, 1,000X HRP conjugated Streptavidin, 2X blocking buffer, wash buffer, detection buffer and cell lysis buffer.
Initially the membrane is blocked for 30 minutes and then incubated with the sample for 1-2 hours. After the washing steps the membranes are incubated with primary antibody for about 1-2 hours (I have tried incubating for just 1 hour and that worked fine). This is followed by the incubation and washing steps; membranes are incubated for about 40 minutes with HRP-conjugated streptavidin and at the end we proceeded with the detection reaction. For convenience the procedure can be done overnight at 4o.
In summary this is a very easy and sensitive method to detect the cytokines in a sample of choice. Instructions are easy to follow and it successfully detected the cytokine profile of my cells of interest before and after treatment with a bacterial toxin.
Jaya Pandey
Research Associate
Developmental Biology Program
Childrens Hospital of Los Angeles
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