I did Western Blot (WB) with Photo-cross linked protein. I had some unusual thing (maybe for me). When i added ECL substrate on membrane. The signals were getting weak with in just 1 mint and eventually completely dropped. What's the possible reason for this and how i can fix it.
Thank you so much for your valuable suggestions.
Yang sangat menarik dengan laser pewarna konvensional, laser yang beroperasi dengan prinsip ini tidak memerlukan sumber laser tambahan secara optik untuk memompa pewarna ke keadaan tereksitasi yang diperlukan, dan mungkin menawarkan keuntungan tambahan dalam hal daya.
Struktur: spektrum ECL sangat dimodulasi oleh struktur perangkat, intensitas keluaran menunjukkan ambang batas yang jelas saat arus penggerak meningkat, dan penyempitan spektral diamati saat intensitas meningkat.
Was it a strong signal? Remember, it's a reaction catalysed by the HRP you've stuck to your membrane, and if you've stuck a LOT of enzyme activity there, the reaction will proceed rapidly in that specific location.
For really intense signals its not uncommon to see "negative" blots, where the background levels of luminescence are visible on the film, while the actual signal is a blank void (having burned out all the ECL reagents before the film can even be applied).
You can fix this by...well, loading less on your gel.
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