Did you do a secondary-only control (no primary)? If not, I recommend it. If you still see the nuclear signal with the secondary, then it is most likely dye-charge binding, where the negatively-charged Alexa Fluor 488 dye is binding to the positively-charged nuclei. Sometimes you see this with mitochondria, too. Standard protein blocking won't stop this, but there is a blocking solution you can use (which would be a blocking step prior to the protein blocking step). It's called the Image-iT FX Signal Enhancer Solution, sold by Thermo Fisher Scientific. It blocks the positive charges.
Did you do a secondary-only control (no primary)? If not, I recommend it. If you still see the nuclear signal with the secondary, then it is most likely dye-charge binding, where the negatively-charged Alexa Fluor 488 dye is binding to the positively-charged nuclei. Sometimes you see this with mitochondria, too. Standard protein blocking won't stop this, but there is a blocking solution you can use (which would be a blocking step prior to the protein blocking step). It's called the Image-iT FX Signal Enhancer Solution, sold by Thermo Fisher Scientific. It blocks the positive charges.
Hi NIco,
have you done the usual routine controls? Omittance of first antibody etc.
Best regards,
Daniela
Hi NIco,
have you done the usual routine controls? Omittance of first antibody etc.
Best regards,
Daniela
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Jason A Kilgore Daniela Rodler
Thanks for your anwsers. I wasn´t aware of the positively-charged nuclei, its a very good tip.
Jason A Kilgore Daniela Rodler
Thanks for your anwsers. I wasn´t aware of the positively-charged nuclei, its a very good tip.
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Did you do a secondary-only control (no primary)? If not, I recommend it. If you still see the nuclear signal with the secondary, then it is most likely dye-charge binding, where the negatively-charged Alexa Fluor 488 dye is binding to the positively-charged nuclei. Sometimes you see this with mitochondria, too. Standard protein blocking won't stop this, but there is a blocking solution you can use (which would be a blocking step prior to the protein blocking step). It's called the Image-iT FX Signal Enhancer Solution, sold by Thermo Fisher Scientific. It blocks the positive charges.
Did you do a secondary-only control (no primary)? If not, I recommend it. If you still see the nuclear signal with the secondary, then it is most likely dye-charge binding, where the negatively-charged Alexa Fluor 488 dye is binding to the positively-charged nuclei. Sometimes you see this with mitochondria, too. Standard protein blocking won't stop this, but there is a blocking solution you can use (which would be a blocking step prior to the protein blocking step). It's called the Image-iT FX Signal Enhancer Solution, sold by Thermo Fisher Scientific. It blocks the positive charges.
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