Thermal Denaturation Assay Services

Target proteins used for crystallization or SAXS can be studied by thermal denaturation assays. The melting point of a protein (Tm) is influenced not only by the nature of the protein itself but also by its chemical environment (e.g. pH, salt nature and concentration, additives, presence of natural or synthetic ligands, etc.). It is possible determine optimum conditions stabilising the protein fold.

Two available instruments to perform thermal denaturation assays:

Thermofluor

In this method, the protein is incubated with a dye that only binds hydrophobic protein regions, where it becomes highly fluorescent. The protein is gradually heated, in order to slowly unfold, exposing these hydrophobic patches.

nanoDSF

NanoDSF is an advanced Differential Scanning Fluorimetry method for measuring ultra-high resolution protein stability using intrinsic tryptophan or tyrosine fluorescence.

Optimization Screening

The RUBIC buffer screen has been designed at the SPC facility and is now distributed under licence by Molecular Dimensions.

Features of the RUBIC Buffer Screen:

• Optimize purification and storage conditions.
• 96 conditions
• Screen for global parameters e.g. pH, salt concentrations, buffer type and concentration.
• Suits a wide range of proteins (small, large, complex, DNA binding proteins etc.) and applications.

Sample requirement for TF:

• ~210ul of protein in low ionic sample buffer free of stabilizing reagent. Initial protein at 20uM (35kDa) is normally sufficient to visualize a melting curve with a good signal-to-noise ratio. Lower concentration can be used with larger protein, while smaller protein may require more concentrated protein
• Sample buffer should contain reagent to stabilize protein, we recommend not to exceed NaCl (<200uM), glycerol (<10%), reducing reagent (<5mM). Assay is not compatible with most of the detergents.

Sample requirements for nanoDSF:

• 210 ul of protein containing 2-3 tryptophans
• Maximal protein concentration (standard IgG) > 150 mgml, minimal protein concentration (standard IgG) 5 ug/ml

Contact SPC to discuss your experiment in detail

Sample optimization using Rubic Additive screen

The additive screen aims to explore the effect of several additives on the thermal stability of the proteins. We recommend to perform this screen to complement the BUFFER screen.

• Monovalent ions, multivalent ions, salts
• Dissociation reagents, chaotropic reagents
• Amino acids, carbohydrates, linker, polyamines, polyols
• Co-factors, nucleotides, chelating reagent, reducing reagents, detergents

Applications:

• Fine-tuning of sample conditining
• Increase protein stability
• Identification of an unkonw ligand partner

Sample requirement for TF:

• 220ul of pure protein at ≈20uM.
• Recommened sample buffer: buffer concentration (<200mM), NaCl (<200mM), Glycerol (<10%) , no stabilizing reagent (DTT, TCEP, etc.), free of detergent.
• 500ul of a 5X buffer (selected from BUFFER screen). Provided by the facility.

Sample requirements for nanoDSF:

• 210 ul of protein containing 2-3 tryptophans
• Maximal protein concentration (standard IgG) > 150 mgml, minimal protein concentration (standard IgG) 5 ug/ml

Contact SPC to discuss your experiment in detail

• Contact the SPC to request thermal denaturation analysis. Indicate the number of samples, screen,  protein MW and concentration, sample buffer composition. You may request a specific date and we will confirm it by email shortly. Sample can be shipped overninght eiher frozen or at 4°C with ice pack (no ice). Please provide tracking number if available.
• The experiment will be performed upon we receive your sample. TF experiment will be carry out using SYPRO Orange and with a temperature ramping from 5°C to 95°C (1°C / min) using a MyIQ RT-PCR instrument (BioRad).
• We will send a short report by email which will contain 1) raw data (CSV) and 2) plotted data using GraphPad (pdf). The SPC can help the researcher with the data interpretation on demand.

MALDI TOF

Determination of the Molecular Mass of your protein sample.

CovalX HM4 High-Mass System enables:

Protein complex analysis
Intact Protein Analysis (upto 2MDa)

Contact us for detailed protocol, instrument booking, training, pricing, etc.