FAQspeak
I am using a reticulocyte lysate-based translation system to express my protein. How can I reduce the background on my Western blots?
When using a rabbit reticulocyte lysate-based translation system to express your protein of interest (e.g., the TNT® T7 Quick Coupled Transcription/Translation System) followed by Western blotting, excessive background can arise on the blot. If the usual issues have been considered (i.e., insufficient blocking or washing, too much lysate loaded onto the gel or blot exposed to film too long), be sure that you are not using a secondary antibody directed against rabbit proteins that will react with the lysate components. If you are experiencing background when using horseradish peroxidase detection, the culprit may be peroxidase activity from the hemoglobin in the reticulocyte lysate. To prevent the hemoglobin from reacting with your substrate during detection, soak the membrane in TBS containing 10% ImmunoPure Peroxidase Suppressor (Pierce Cat.# 35000) for 30 minutes prior to blocking.
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