Titration assays were performed to determine the functional protein concentrations of the LRRK2 variants using the high-affinity inhibitor MLi-2 (Merck, U.S.A.). Therefore, 24 µl of Buffer I (25 mM Tris–HCl, 50 mM NaCl, 20 mM MgCl2, 1 mM GDP, 1 mM DTT, 0.5 mg/ml BSA, 52.1/104.2 nM LRRK2 [total protein concentration after Bradford were mixed with 1 µl of a MLi-2 dilution series (50× concentrated) prepared in 100% DMSO [26 (link)]. The reactions were started by mixing 10 µl of this reaction mix with 10 µl of Buffer II (25 mM Tris–HCl, 50 mM NaCl, 1 mM DTT, 0.5 mg/ml BSA, 1900 µM LRRKtide, 100 µM Fluorescein-LRRKtide, 300 µM ATP) in a 384 well-plate and monitored for 60–90 min with a LabChip EZ Reader (PerkinElmer). The resulting conversion rates were plotted against the respective MLi-2 concentrations and the x-axes intersection of the respective linear fit was determined to obtain the functional protein concentrations using GraphPad Prism 6 (assuming a 1 : 1 binding of MLi-2).
Mli 2
Manufactured by Merck Group
Sourced in United States
The MLi-2 is a laboratory equipment product manufactured by Merck Group. It is designed to perform a core function within the laboratory setting. This factual and unbiased description is provided without any interpretation or extrapolation on the intended use of the product.
Automatically generated - may contain errors
Lab products found in correlation
Labchip ez reader, by PerkinElmer (1 mentions)
Mli 2, by GraphPad (1 mentions)
Prism 6, by GraphPad (1 mentions)
Gsk2578215a, by Bio-Techne (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
D01060501, by Research Diets (1 mentions)
Dyenamic et terminator chemistry, by Cytiva (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
6 protocols using mli 2
1
LRRK2 Variant Functional Concentration Assay
2
Obtaining and Characterizing Small Molecule Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GSK2578215A (Reith et al, 2012 ) was obtained from Tocris or GlaxoSmithKline; MLI‐2 (Fell et al, 2015 ) was obtained from Merck; Bafilomycin A1 (#B1793) was obtained from Sigma, and HG‐10‐102‐01 (Choi et al, 2012 ), LRRK2‐IN1 (Deng et al, 2011 ) and Vps34‐IN1 (Bago et al, 2014 ) were custom synthesised by Natalia Shapiro (University of Dundee, UK).
3
Investigating LRRK2 Kinase Inhibition in Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zymosan A from Saccharomyces cerevisiae (Sigma, St. Louis, MO, USA, #Z4250) and MLi-2 from Merck (Tocris, Bristol, UK, #5756) were used for these experiments as TLR2 inflammatory stimuli and an LRRK2 kinase inhibitor, respectively. For immunoblots, primary microglia from wild-type and G2019S-LRRK2 Tg mice were treated with 200 µg/mL zymosan and 1 µM MLi-2 for 4 h and 24 h, and for RNA-Seq, they were treated with the same concentration of zymosan and MLi-2, but for 24 h only. Zymosan dose was chosen based on previous studies that have used 200 μg/mL concentration of zymosan in immune cells, such as in BMDMs [16 (link),20 (link)]. For MLi-2, previous studies have used a 1 mM concentration of MLi-2 in vivo [21 (link)] and a 0.5 mM concentration of MLi-2 in vitro [22 (link)].
4
Detailed Reagent Acquisition Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GSK2578215A was obtained from GlaxoSmithKline [29 (link)]. HG-10-102-01 was custom synthesized as described previously [30 (link)]. MLi-2 was obtained from Merck and also synthesized as described in [31a ]. Phos-tag acrylamide was synthesized as described in [31b ]. Phos-tag acrylamide was stored at 5 mM aqueous solution (3.43 mg of compound in 1 ml of solution) at 4°C in black tubes that block out light as Phos-tag acrylamide is light-sensitive. HPLC analysis of stock Phos-tag acrylamide was undertaken every 4–5 weeks to ensure stock had not started to polymerize. All recombinant proteins, DNA constructs and antibodies generated for the present study can be requested via our reagents website (https://mrcppureagents.dundee.ac.uk/ ).
5
Synthesis and Formulation of MLi-2 Diet
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MLi-2 was synthesized at Merck & Co., Inc. (Boston, MA). Diet was prepared at Research Diets (New Brunswick, NJ), and formulated to deliver a dose of 0, 10, or 60 mg/kg MLi-2 per day, based on an average daily food consumption of 4 g per 30 g mouse. The base for each diet was the vehicle diet (D01060501; Research Diets).
6
Characterization of LRRK2 Kinase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[γ-32P] ATP was from PerkinElmer, MLi-2 was obtained from Merck [12 (link)]. LRRK2 (G2019S) Recombinant Human Protein (residues 970–2527 #10499963) was purchased from Invitrogen and all additional kinases used in the in vitro screen were from MRC PPU Reagents and Services — including TAK1 (1–303), TAB1 (437–504) fusion (DU753) and N-terminal GST-MST3 (DU30889). All mutagenesis was carried out using the QuikChange site-directed mutagenesis method (Stratagene) with KOD polymerase (Novagen). All DNA constructs were verified by DNA sequencing, which was performed by The Sequencing Service, School of Life Sciences, University of Dundee, using DYEnamic ET terminator chemistry (Amersham Biosciences) on Applied Biosystems automated DNA sequencers. DNA for bacterial protein expression was transformed into E. coli BL21 DE3 RIL (codon plus) cells (Stratagene). All cDNA plasmids, antibodies and recombinant proteins generated for this study are available to request through our reagents website https://mrcppureagents.dundee.ac.uk/ or from the Itzen laboratory.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!