Hi All,
I have some questions on the thermal stability shift assay.
1. what is the ideal hold time for thermal scanning of protein? higher molecular weight needs more holding time whereas lower molecular weight protein needs less. is it so? I know that hydrophobic segments present in the proteins determine the intensity of RFU and the compactness of the protein. If the full- length protein needs 3 mins hold- time, could it be feasible to think that N terminal and C terminal fragment of the protein should be working in a shorter hold time?
2. what is the ideal stoichiometry when calculating the ideal working concentration of the dye and protein?
3. Does the Lid temperature have any negative impact on sample temperature?
4. what should be the ideal working volume of performing thermal -shift?
5. If I have two proteins and want to see whether the presence of the first protein has any regulation on the second protein, can we do that in thermal stability assay?
Dear Abir
Here some answer to your questions based on my experience:
1. What do you mean for hold time? What is critical for DSF in the scan rate because if you scan to fast you will have late response of the sample and you curve will be shifted.
Generally 1°C/min it is a good scan rate. I do not think that there is any relationship between MW and it.
If you are afraid that the kinetic of your protein is very slow you can just run 2 experiments with different scan rate and see if the curve change or not. If increasing the scan rate the melting curve of your protein translate versus lower temperature than you need to adjust it.
2. Generally considering the Sypro Orange show low fluorescence if the protein is folded, we use an excess of it (50X final concentration) and a sample concentration of about 5-20uM depending of its MW.
3.I do not think so. Sample generally do not feel the lid temperature. It is done just to prevent sample evaporation.
4. what should be the ideal working volume of performing thermal -shift?
Generally i'm using 40ul total volume 4ul of SYproOrange 50X plus 36ul of protein sample. Pay attention to the bubble and perform replicates.
5.It is difficult. You can use it to see interaction of a proteins with small molecules or peptides that do not bind sypro orange, but if you have 2 proteins, Sypro orange may bind to both protein and on the complex and your profile is the resultant of several unfolding processes (especially if the protein ratio is not exactly 1:1).
To obtain something clear you need to be in the lucky case that all the protein are involved in the complex and that thethe interaction of the 2 proteins increase a lot the melting temperature of both and therefore you will se a strong shift.
However if you have samples, sypro Orange and a RT-PCR machine, it is fast and simple to try! Remind to run in parallel all the controlls as single proteins and buffers with out proteins.
Actually i'm preparing a presentation about DSF to load on my blog: ProteoCool ( https://proteocool.blogspot.com/ ) that may contain some examples usefull to you. I hope to be able to finalize it and upload it on the blog before the end of October.
good luck
Manuele
Dear Abir
in case you are still interested, i uploaded a dsf presentation on my blog: ProteoCool ( https://proteocool.blogspot.com/ )
at page3.
i hope it could be usefull to you.
good luck
Manuele
- Badges
- Science topic