Hi
I am quite new to this technique and so would require some help. I am trying to quantify the binding of a ligand to a protein using thermal shift assay. I am using Sypro Orange Dye to bind to the protein. I am trying to optimize the protein: dye ratio and have tried the following conditions so far:
1X Dye, 2.5X Dye, 5X Dye and 10 X dye and protein concentrations ranging from 300 nM to 7 uM. The buffer I am using is Tris (pH 7.6) and 150 mM NaCl. However, in all the cases, I am getting a high background signal (buffer + dye only), which is comparable to that when low protein amount is present. What could be the possible reason for this high background fluorescence.
On another note: I am trying it in triplicates and am getting high variability in the signals (Although the trend is consistent). How can I reduce this high variability
Dear Archishman Dakua
theoretically sypro orange at the right concentration (generally i'm using the
https://www.sigmaaldrich.com/IT/en/product/sigma/s5692
or
https://www.thermofisher.com/order/catalog/product/S6650
diluter 1000times (5X final concentration)
To avoid pipetting of too small volumes that can cause sample to sample variability i'm preparing an intermediate 50X stock (by diluting 100 times the original stock and then i'm mixing 4ul of the 50X Syproorange stock with 36ul of the protein solution directly in the multiplate suitable for RT-PCR.
Is this similar to your protocol? IF not, which are the differences?
if you are using this range of syproorange concentration, high background and well to well variability is strange is a simple Tris,NaCl and i suspect that there is a issue in some of your reagents or in the plate assembly procedure (eg presence of bubbles)
best regards
Manuele
Hi Manuele Martinelli
I am using a similar protocol. I am preparing an intermediate 50X stock . However, I am adding the Sypro directly to the protein in an eppendorf and mixing. After that I am adding equal volumes to the multiple .
I think there might be bubbles present during pipetting and I'll try to make sure that doesn't happen.
Also can the Tris NaCl buffer cause high background fluorescence? Will changing buffer to HEPES or PBS work?
Thank you for your help
Regards
Archishman
Dear Archishman Dakua
i do not thin kthat the problem is due to the Tris buffer since i'm using it for DSF and i do not have any problem.
Are you sure that you plate and sealing film are compatible with RT-PCR? since sometimes plastic of low quality used for standard PCR can be not really trasparent and you observe fluorescence background due to light scattering trough the plastic.
IS you Syproorange stock new? or old?
best regards
Manuele
Hi Manuele Martinelli
I am using Microseal B as recommended by Biorad and was using clear plates for the RT-PCR.
The Sypro Orange stock is completely new and I dilute it fresh every time before my experiment.
I am attaching a representative figure of my experiment. As you can see in the case of buffer + dye , there is a peak occurring at around 60 degrees, which is not present when there is buffer only (I did a negative control to check whether there is any background fluorescence from the plate).
The signal from the protein is significantly higher than the background, but I'm still concerned about that peak from Sypro Orange and would like to have an optimal experiment where I don't have that peak. However if it's a property of Sypro Orange then I don't think I can do anything about it.
Any help in this regard would be greatly appreciated.
Thank you
Regards
Archishman
I see the same thing with a big peak with sypro orange, no protein.
I switched plates and the peak went away. Biorad plates gave a peak, USAScientific plates did not
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