We have received some questions about primary and secondary antibodies when doing chemiluminescent Western blots that have been frequently asked by customers.
Below are a few of the most common questions asked.
Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band.
Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.
Does it matter where I purchased the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. The ratio of HRP enzyme to antibody varies as well, and may affect the detection of the target.
If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.
LI-COR offers high-quality secondary antibodies for chemiluminescent Western blotting that have been optimized for use with WesternSure® PREMIUM Chemiluminescent Substrate.
Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.
What questions do you have?
Chemiluminscent Western Blot FAQs goes through other frequently asked questions we have received from customers.
And, if you want to optimize your chemiluminescent Western blot, Optimizing Chemiluminescent Western blots Technical Note might be a good place to start.
If you want to save time and money by going digital - and have access to near-infrared fluorescence detection - explore the advantages of the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System.
If you have other questions not covered in these technical notes, contact us. Our experts are standing by through email, phone or chat!
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