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Protein stability and aggregation assay kit

BHE16300388

This product includes 60 ul of 1000 x PSA dye for more than 200 assays with the assay volume of 0.3 ml using 96-well plates or cuvettes.

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+1 866.986.9598
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[email protected]
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Reference 1. Takahashi N et al, TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P₂. Nat Commun. 26;5:4994 (2014).
2. Srinivasan S. et al, Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis. Angiogenesis 18:449–462 (2015).
3. Matsuura, S et al; Enzyme Immobilization in Mesoporous Silica for Enhancement of Thermostability. Journal of Nanoscience and Nanotechnology, Volume 18, Number 1, pp. 104-109 (2018). 
Product Manual Datasheet
Protocols Description PROTOCOL - Protein Thermal Unfolding Curve and Protein Stability
The protein sample can be a purified protein or a protein mixture. The protein solution and buffer should not contain any detergent. Most proteins unfold at a temperature between 50 and 100 °C within 15 min.
Select an appropriate temperature for the unfolding reactions to complete in the time scale of 5 min to 30 min for the convenience of measurement.
1. Prepare 1 ml of 0.5 mg/ml protein solution. Divide the protein solution evenly in 9 aliquots (110 µl each). Keep the samples on ice.
2. Keep one sample on ice as a time zero control. Incubate the other 8 samples in a water-bath at the selected temperature and start to record the incubation time. Move one sample from the water-bath to ice at each time point until all the samples are collected. Transfer 100 µl of each sample into the wells of a black 96-well plate.
3. Mix 3 µl of the 1000 x PSA dye with 3 ml of water to make the 1 x PSA dye. Add 200 µl of the 1 x PSA dye into the wells with the protein samples. Incubate the mixtures at room temperature for 5 min.
4. Read the fluorescence intensity at 610 nm (excitation at 550 nm).
 
Data Processing
Calculate the half life time t1/2 using the following curve fitting formula:
 
                                                   Fluorescence change ΔF = F - FN = [FU x t / (t1/2 + t)]
 
where t is the incubation time of the protein at the selected temperature; F is the fluorescence of the sample at time t; FN is the fluorescence of the unheated sample (t = 0); t1/2 is the half life time; FU is the maximum fluorescence of the curve representing the fluorescence of the completely unfolded (denatured) protein. The F, FN and t values are from the measurement. The t1/2 and FU values are obtained from the fitting. Standard curve fitting programs for binding Kd or enzyme Km calculation can be used to calculate the t1/2 and FU values.
 
Protein Unfolding Aggregation State
An authentic native protein sample is needed to read the background signal. All samples are prepared at the same protein concentration in the same buffer. The buffer should not contain any detergent. The measurement is performed at room temperature.
1. Prepare 100 µl of native protein solution, 100 µl of sample protein solution and 100 µl of unfolded protein solution. The unfolded protein solution is prepared by heating.
2. Dilute 1 µl of the 1000 x PSA dye with 1 ml of water to make the 1 x PSA dye. Mix 200 µl of the 1 x PSA dye with 100 µl of the protein solution for 5 min. Read the fluorescence at 610 nm (excitation 550 nm). Record the fluorescence values:
                                                  Sample                               Fluorescence
                                                  Native protein                    FN
                                                  Unfolded protein               FU
                                                  Sample protein                  FS
3. Calculate the percentage of the unfolded protein in the sample protein using the following formula:
 
                                                   Unfolded (%) = 100 % x (FS – FN) / (FU – FN)
 
Control Proteins for Unfolding Aggregation Reaction
Commercially available proteins such as lysozyme (Sigma Catalog No. L6876) or lactate dehydrogenase (LDH, Boeringer product) at a concentration of 1 mg/ml can be used as controls. BSA is not a good control.
SKU Supplier No. Size Your price Quantity
BHE16300388 PSA200K 200 assays $140.38
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