Good working final concentration of protein is 150-350ng/ul.

You can prepare stock of SYPRO 1:200 diluted in water and 5x final concentration usually works fine with protein concentration indicated as above.

For me your sample is either already denaturated (agree with Brian) or has a lot of aggregates! (which give an abnormal initial signal). Never forget to centrifuge protein samples before Thermal shift assay.

When you do serial dilutions of the SYPRO, does the initial signal track? i.e. if you double the SYPRO from 1X to 2X, does the initial fluorescence also double? The graph of your data looks like the signal may be saturated to begin with.Did you double check that you are using the correct filter?

Check the initial signals with and without protein also. I high initial signal could indicate that your protein is not stable in these conditions and has already unfolded prior to increasing temperature.

You may also want to try a control with something like lysozyme to narrow down whether its your protein or some technical problem.

Good working final concentration of protein is 150-350ng/ul.

You can prepare stock of SYPRO 1:200 diluted in water and 5x final concentration usually works fine with protein concentration indicated as above.

For me your sample is either already denaturated (agree with Brian) or has a lot of aggregates! (which give an abnormal initial signal). Never forget to centrifuge protein samples before Thermal shift assay.

I would suggest, try using buffers such as PBS or HEPES (but not Tris). Protein concentrations to be used would depend on molecular mass of your protein but for of proteins (10-20 kDa), 2-4 microMolar is sufficient. I noticed that sometimes when the stability of protein is less , then you may need little more protein in the assay. About 2-4X sypro is sufficient for the assay. If you use higher amount of that you may get non -specific signals too.  Try diluting Sypro (I always used from Sigma) in the assay buffer. I hope this works for you.

hi,

i am also working on lipase of same molecular weight, here i used buffer with out Glycerol and EDTA. when i use 5% glycerol it is giving high background florescence, and its same if i add high amount of Sypro orange. finally i optimized to use  20 nM of protein in Tris buffer pH-8 with 1X sypro orange dye.

(Dye concentration depends on amount of protein you are using and composition of hydrophobic residues in your protein) 

hope this will work for u. 

There are some proteins that do not work well with hydrophobic dyes such as Sypro Orange.

You might want to try nanoDSF instead which measures thermal and chemical stability of proteins by detecting the change in tryptophan fluorescence upon unfolding. Since no dyes are required, you can even work with hydrophobic proteins, such as membrane proteins, and in the presence of detergents!

Follow this link to learn more about nanoDSF

http://www.nanotemper-technologies.com/products/prometheus-series/prometheus-nt48/

I am using SYPRO Orange Dye for BSA. and the protein concentration is around 20mg/mL. I got the same results as you did. So I need help too..

rajsee devraj pawar, 20mg/ml is too high. use 1 to 2mg /ml. also try low concerntration of imidazole, which will help in solubility of protein.

As mentioned in AB documents, glycerol or detergents may cause high background of fluorescence signal. 

One thing you should take into account is that the temperature of the machine that you start running the assay.  

I would recommend that you spin your samples at top speed in a bench-top centrifuge prior to loading them into the microplate.

High initial signal could be indicative that your protein is already unfolded, and the subsequent increase is hydrophobic patches being buried due to aggregation. Have you tried CD-spec to see what secondary structure there is present?

You should try running a commercial protein in the same assay to confirm that your protein sample is the problem. Remember, it's all about the controls!

I would also like some recommendations. My protein of interest is a monomer at 4C and forms nano particles at higher temps. We can see this using circular dichroism, however, I'd like to try to measure fluorescence with Sypro Orange. The stock is 5000x - however I've seen papers use that 5000x stock differently. Firstly, dilute the 5000x stock to 40x stock in PBS. then use 2.5 ul of the 40x stock in final volume of 20 ul with your protein of interest. Paper took readings every 5 sec at 5, 15, 25, 35 C, and fluorescence increased as a function of urea with protein... makes sense. I'll try this approach... has anyone found a simple way to interpret the data?

May I add a question to this thread? Is there a molecular weight cut off of proteins compatible with TSA? I have an NTD of a protein consisting of 3 alpha helices which is only 66 residues and I fear it may be too short to allow detectable unfolding. Can anyone offer advise or guidance on the subject? Thanks.

@ Anna Belorusova - Can the SYPRO orange dye be diluted in water/buffer and stored at -20 or 4 degrees for a period of 1-3 months? I do not want to use DMSO as it interferes with my assay.

Thanks

@ Jyoti chhibber-Goel, No the dye is so sensitive that when you dilute it in water/buffer it loose its activity as early as 2 or 3 days. So it ideal to make a fresh dilutions whenever it is needed. Instead you can aliquot and use to prevent frequent freeze thaw.

Vishweshwar kumar Ganji Do you know why the dye degrades in aqueous condition?

Sorry @sayeeda chowdhury I am unaware of the mechanism behind that. But, based on my work experience, I noticed a great difference between freshly prepared stock and stored stock of sypro orange dye.