Monolith protein labeling kit red nhs 2nd generation

Protein-protein interactions were measured using microscale thermophoresis on a Monolith 1.15 (Nanotemper Technologies, Munich, Germany). Measurement settings: LED power at 30%, laser power at 75%, temperature fixed at 21 °C. (NIA)₂ and variants were labelled according to manufacturer’s instructions (Monolith Protein Labeling Kit RED-NHS 2nd Generation, MO-L011, Nanotemper Technologies). 200 nM labelled protein was titrated with a 1:1 dilution series of wild-type ISCU2 or the respective variant starting from 100 µM to 200 µM as indicated. Data were analyzed using Origin 8 G (OriginLab Corporation, Northampton, MA, USA).

Microscale thermophoresis was performed by using Monolith NT.115 (Nano Temper Technologies, Munich, Germany), as previously described [52 (link)]. Proteins were labelled using the Monolith Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies, Cat.-No. M0-L011). The compounds were diluted in 16 different concentrations ranging from 6.1 nM to 200 µM, and labelled SARS-CoV-2 NP NTD (c = 3475 nM), labelled SARS-CoV-2 NP (c = 880 nM), labelled SARS-Cov-1 NP (c = 670 nM), or labelled SARS-CoV-2 omicron variant NP (c = 250 nM), respectively, were added 1:1. MST-Buffer (50 mM Tris-Base, 150 mM NaCl, 10 mM MgCl₂ and 0.05% v/v Tween 20, filled up to 100 mL H₂Odest.) was used for the dilution of the target proteins and the microscale thermophoresis preparations. The samples were incubated for 30 min in the dark and measured using the Monolith NT.115 instrument [53 ]. LED-power was set on 30% and MST-power on 10% for recombinant SARS-CoV-2 NP wildtype and omicron variant and SARS-CoV-1 NP, respectively. For recombinant SARS-CoV-2 NP NTD the LED-power was set on LED-power 40% and MST-power on 60%. Fitting curves with Kd values were calculated with MO.Affinity analysis software (NanoTemper Technologies, Munich, Germany).

The MST assay was performed as previously described with minor modification.^{60 (link)} The affinity of the purified ALR1^CD or its mutant (ALR1^CD4C-A) with AlCl₃ or other metal ions (LaCl₃, CdCl₂, CeCl₃, InCl₃, GaCl₃, FeCl₃, MnCl₂, and CaCl₂) was measured using the Monolith NT.115 (Nanotemper Technologies). Proteins were first fluorescently labeled using the Monolith Protein Labeling Kit RED-NHS 2nd Generation (Nanotemper Technologies, MO-L011) according to the manufacturer’s protocol, and the labeled protein used for each assay was about 100 nM. A solution of unlabeled metal ions was diluted for appropriate serial concentration gradient. The samples were loaded into MST standard capillaries (Nanotemper Technologies, MO-K022). Measurements were performed in buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 2 mM DTT, and 0.05% Tween-20, by using medium MST power and 20% LED power. Data were fitted in Kd model using MO.Affinity Analysis v2.2.4, and were finally displayed in ΔFnorm normalization.

The dissociation constant (Kd) between the W25 Nanobody and SARS-CoV-2 S1 of Spike RBD was measured by microscale thermophoresis (MST) using a Monolith NT.115PICO instrument (Nanotemper Technologies). Purified W25 was buffer exchanged into a PBS buffer, pH 7.4, and its concentration was adjusted to 10 µM using UV-absorbance. Next, W25 was fluorescently labeled with the Monolith Protein Labeling Kit RED—NHS 2nd Generation (MO-L011, NanoTemper Technologies) following the protocol established in the manual. Labeled W25 was centrifuged at 14,000 rpm for 15 min to eliminate precipitates. A 16-point serial dilution series of recombinant Spike RBD (250 nM to 7.6 pM) was applied in PBS buffer containing 0.01% Pluronic F-127 and mixed with a final concentration of 1 nM labeled W25. Affinity measurements were conducted in Premium Capillaries Monolith NT.115 (MO-K025, NanoTemper Technologies) and repeated three times. Recombinant SARS-CoV-2 (2019-nCoV) Spike S1 Protein (RBD) from Trenzyme, Germany was used for the assays.

The CatD of ENB1 or ENB1^G780R (enb1), as described previously with minor modifications (Olek et al., 2014) (link), was used for the UDP-Glc (Sigma-Aldrich, St. Louis, MO, USA; cat. no. U4625) binding assay. ENB1 or enb1 CatD corresponding to the 1,015–2,523-bp region of ENB1 or enb1 ORF was cloned into the pCold-TF DNA Vector (Takara, Shiga, Japan) that contains an N-terminal His tag and a soluble tag of trigger factor (TF) chaperone. The recombinant His-TF-ENB1 CatD or His-TF-ENB1^G780R CatD was expressed in E. coli Rosetta (DE3) cells by adding 0.5 mM of IPTG when OD₆₀₀ reached 0.8. Recombinant proteins were purified with the BeaverBeads IDA-Nickel (Beaver, Suzhou, China; cat. no. 70501-5) and then labeled with the Monolith Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper, Munich, Germany; cat. no. MO-L011). The microscale thermophoresis assays were conducted using a Monolith NT.115 (NanoTemper, Munich, Germany) machine. The serial concentrations of UDP-Glc were titrated against 100 nM of His-TF-ENB1 CatD or His-TF-ENB1^G780R CatD protein in the optimized buffer (50 mM Tris–HCl, pH 7.4, 150-mM NaCl, 10-mM MgCl₂, 0.05% Tween-20). Each protein was labeled 3 times for three independent tests. All data were analyzed using the MO. Affinity Analysis version 2.3 software. Relevant primer sequences are given in Supplemental Data Set 6.

Microscale thermophoresis (MST) was performed for assessment of the interaction between compound (5) and AURKA (Sigma-Aldrich, Germany). The method is performed as previously described [31 (link), 32 (link)]. The AURKA protein was labeled using Monolith Protein Labeling Kit RED- NHS 2nd Generation (MO-L011, NanoTemper Technologies GmbH, Munich, Germany) according to manufacturer’s instructions. The AURKA protein concentration used was 400 nm, it was titrated against different concentrations of compound (5). Analysis buffer used includes; 50 mM Tris buffer pH 7.0, 150 mM NaCl, 10 mM MgCl₂ and 0.05% Tween 20. Samples of interaction were filled into Monolith NT.115 standard capillaries (MO-K022, NanoTemper Technologies GmbH, Munich, Germany). Monolith NT.115 instrument (NanoTemper Technologies) was used for fluorescent signal measurement. Test was performed using 60% LED power and 40 MST power. For analysis, we used MO.Affinity analysis software (Nano Temper Technologies) to generate of fitting curve of interaction and calculation of dissociation constant (Kd).

Microscale thermophoresis (MST) was performed for assessment of the interaction between sertraline, thioridazine, and recombinant human TCTP. The method was performed as previously described (Fischer et al, 2021a (link); Fischer et al, 2021b (link)). The TCTP protein was labeled using Monolith Protein Labeling Kit RED- NHS 2nd Generation (MO-L011, NanoTemper Technologies GmbH, Munich, Germany) according to the manufacturer’s instructions. TCTP protein at 785 nM was titrated against different concentrations of sertraline and thioridazine. The following analysis buffer was used: 50 mM Tris buffer pH 7.0, 150 mM NaCl, 10 mM MgCl₂ and 0.05% Tween 20. Samples of the interacting components were filled into Monolith NT.115 standard capillaries (MO-K022, NanoTemper Technologies GmbH, Munich, Germany). Monolith NT.115 instrument (NanoTemper Technologies) was used for fluorescent signal measurement. The test was performed using 20% LED power and 10 MST power. For analysis, we used MO. The fitting curve of interaction and calculation of dissociation constant (K_d) was performed with Affinity analysis software (Nano Temper Technologies).