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Thanks for replying… I’ve attached the detailed procedure I used.
Asked on Sep 12 2012
Thank you for these additional details.
Your experimental set up is good and you have done a nice job of following the steps and setting the experiment up correctly. Wecan’t say why these samples at very high concentration areincreasingthe signal – an artifact – but they do. Decreasing the sample concentration to account for this artifact is not an option because the degree of nitration is null or too low to measure after dilution so it’s a bit of a catch 22.
It does appear that there is not asignificantamount of nitration labeling in either sample. However, at this point we can’t say exactly what’s going on with these samples so we are happy to offer a refund or replacement product to you. Our lab has suggested a nitration antibody for Western blotting.
When doing Western blotting you should be very careful with highly concentrated serum samples to prevent non specific binding due to large protein concentration in samples (so do significant dilution of SDS PAGE sample) or presence of human IgG in sample.
I look forward to hearing how you would like to proceed. If you could please provide yu Abcam order confirmation number I will be able to process either the replacement or refund for you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on Sep 12 2012