I know that SYBR-safe can stain ssDNA and RNA with less efficiency than dsDNA, but can it stain also single nucleotides as ATP/GTP?
I'm asking this question as I've purified a pure GST-tagged GTPase (SDS checked) and the absorbance peak is sharp at 260 rather than 280 (my protein is not known to bind DNA). I've then tried to eliminate the presumed contaminant DNA by anion exchange DEAE column and the spectrum of the elution is still the same. I've then loaded the protein sample on agarose gel colored with SYBR-safe and an intense band appeared.
Can the brightness be due to the bound GTP rather then DNA? And therefore, what's the capacity of SYBR-safe to bind single nucleotides?
Thank you all
Hi Valerio,
I highly doubt that SYBR safe (or other interchelating agents dedicated for nucleic acid staining) could stain free nucleotides. If this was the case, anyone trying to gel purify the PCR product would witness intense fluorescence resulting from excess dNTP. I also routinely prepare adenylated adaptor on my own, which involves incubation of adaptor with excess amount of ATP followed by gel purification on 20% PAGE. 20% PAGE is capable of resolving free NTPs (which I confirmed with 32P-ATP), but I have never seen any fluorescence from the residual ATP at the expected position after staining with SYBR Gold.
To clarify the identity of the intense band you observe, I would suggest digesting your protein prep with proteinase K and resolve the product on an agarose or polyacrylamide gel with size markers.
Hi Valerio,
SyBR safe is an intercalating agent which is specifically designed to insert between bases in strands of DNA. So in theory there should be no way for it to stain single nucleotides.
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