鸡IgY,多克隆抗体-同型对照(BSA and Azide free) (ab37382)

Thank you for contacting us. Unfortunately, I would not recommend using a chicken antibody against a different target (such as ab1394 or ab63504), as it cannot be guaranteed that they do not react with any human antigen. I would rather suggest to use a chicken IgY polyclonal isotype control, as these isotype controls are specifically tested not to cross react. As such we currently have ab37382 available (Click here (or use the following: https://www.abcam.com/Chicken-IgY-polyclonal-Isotype-Control-BSA-and-Azide-free-ab37382.html).). As this antibody however is not labelled with FITC, I may recommend our EasyLink Conjugation kit (ab102883 - ab102885, see below and attached). For the compensation and set-up of the flow cytometer, I would suggest to use ab63498 to ensure the optimal settings. EasyLink antibody conjugation kits: https://www.abcam.com/easylink Click here (or use the following: https://www.abcam.com/EasyLink-FITC-Conjugation-Kit-1-x-1mg-FITC-ab102883.html). Having said this however, I may let you know that isotype controls do not really represent a good negative control. The reasons become apparent in your particular case: The control antibody is of a different preparation ('chicken', or in case of monoclonals a different clone) and therefore may show varying binding properties compared to the specific antibody of interest (ab63498). Furthermore, as the conjugation conditions may have been different, the labelling with the fluorochrome can vary which can be a very important issue in flow cytometry. As a result of different antibody-to-fluorochrome ratios and/or different fluorochrome batches, the control antibody might indeed show a very different staining pattern, and even if it does not bind at all (as it is supposed to) can for these reasons not directly be compared to the antibody of interest. Instead, in flow cytometry another type of control is often used to determine positivity for the marker of interest. This control is termed fluorescence minus one or FMO and works like an isotype control without the actual antibody. FMO controls are set up by leaving out one of the antibodies in your staining panel. You may find more information about this type of control in the latest publications or on flow cytometry websites, such as http://www.flowcytometryuk.org/, http://www.cyto.purdue.edu/ or http://www.isac-net.org . Plus, your local flow cytometry community (core facility?) might be happy to help and advise in the use of optimal controls which are required for publishing the results. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your reply. A549 cells were recommended because they showed weak to no staining using a similar antibody.  Plus these are the results found in the Human Protein Atlas: http://www.proteinatlas.org/ENSG00000169252/cell/A-549 I'm not sure if I have a clear explanation as to why these stained well because fewer than 5% were shown to have a signal in the Human Protein Atlas, unless the antibody was binding to something non-specifically.  Did you run a no-primary control?   Let me know how the staining goes with the mouse keratinocytes.  I look forward to your reply.

Thank you for your reply and for providing the requested information. After seeing the data and your protocol, I would like to make some suggestions that may improve your results. One of my colleagues who has worked with chicken antibodies in the past has found them to be a bit "sticky" and suggested the following: Increase the amount of serum and blocking time. I see that for non-permeabilized cells you use 10% serum - I would advice increasing to 10% and incubating for 1-2 hours. Titrating the antibody concentrations - You appear to get a nice specific signal at 1:250. Continue to titrate that so that you still observe the specific signal and reduce the non-specific signal from the isotype control. Overall, the background is not too bad and this should be easy to titrate. I have contacted the laboratory regarding a negative control and will forward that information to you as soon as possible. I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.