Full-length inpp5ka (NM_001089493.1) and inpp5kb (XM_021479346.1) cDNAs were generated by gene synthesis and cloned into the pGEX-1 to generate GST-fusion proteins (Genewiz/Azenta Life Sciences). GST-human INPP5K and GST were used as positive and negative controls respectively (Weissner et al. 2017 (link)). Constructs were transformed into BL21 DE3 pLysS, induced with 100uM IPTG overnight, and harvested by centrifugation. Cells were lysed in assay buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl2) plus 1% Triton X-100, EDTA-free protease inhibitors (Roche Diagnostics) and turbonuclease (Sigma). GST fusion proteins were affinity purified over gluthione sepharose 4B (GE Healthcare). After extensive washing, aliquots of beads were run on Coomassie gels to determine the abundance of full-length fusion proteins. Beads bearing equal amounts of fusion proteins were incubated in assay buffer containing 135 μM PtdIns(4,5)P2diC8 or PtdIns(3,4,5)P3diC8, including control wells with no enzyme or no substrate lipid, and incubated for 1 h at 37C. Free phosphate was measured using the Malachite Green assay kit (Echelon Biosciences). Results of three independent experiments were presented as mean ± standard deviation. To minimize variability between purifications, all constructs were freshly prepared and purified in parallel for each experiment.
Malachite green assay kit
Manufactured by Echelon Biosciences
Sourced in United States
The Malachite Green Assay Kit is a colorimetric assay used to quantify inorganic phosphate (Pi) in biological samples. The kit utilizes the reaction between malachite green, molybdate, and Pi to produce a colored complex that can be measured spectrophotometrically.
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Lab products found in correlation
Dic8 ptdins 4 5 p2, by Echelon Biosciences (2 mentions)
Edta free protease inhibitor, by Roche (1 mentions)
Turbonuclease, by Merck Group (1 mentions)
Malachite green phosphatase assay kit, by Echelon Biosciences (1 mentions)
Protein a sepharose beads, by Merck Group (1 mentions)
Rabbit anti pten antibody, by Cell Signaling Technology (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Edta free protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Dic8 ptdins 3 4 5 p3, by Echelon Biosciences (1 mentions)
Gfp trap ma beads, by Proteintech (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gfp trap beads, by Smart-Lifesciences (1 mentions)
9 protocols using malachite green assay kit
1
Purification and Characterization of INPP5K
2
PTEN Phosphatase Activity Assay
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The phosphatase activity of the PTEN was determined using the Malachite Green Assay Kit (Echelon Biosciences, Salt Lake, UT, USA) [43 (link)]. Briefly, the recombinant PTEN enzyme (Echelon Biosciences) was reconstituted in distilled water to yield a 50 μg/mL concentrate. Phosphatidylinositol 3,4,5-trisphosphate diC8 (PIP3) was purchased from Echelon Biosciences. Each reaction (final volume of 25 μL) contained 75 ng PTEN enzyme and 3 nmol of PIP3 in the PTEN reaction buffer (25 mM Tris–HCl pH 7.4, 140 mM NaCl, 2.7 mM KCl, 10 mM DTE). After incubation at 37 °C for 60 min, 100 μL of malachite green solution (Malachite Green Phosphatase Assay Kit, Echelon K-1500) was added. After 20 min of development of the color at room temperature, the absorbance at 620 nm was measured. The phosphate standards (Echelon K-1500 kit) were used to create the standard curve to quantify phosphate production. The relative phosphate amount to the control (i.e., 0 μM of MI) was calculated and represented as the PTEN activity.
3
PTEN Phosphatase Activity Assay
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MM and ULM cells were cultured in 100-mm dishes in complete DMEM-F12 1:1 medium until 80 to 90% confluence. Cells were lysed in ice-cold 25 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 5% glycerol lysis buffer. PTEN (500 μg) was immunoprecipitated from whole-cell lysates with 8 μl of rabbit anti-PTEN antibody (Cell Signaling Technologies) under agitation at 4°C overnight. The antigen-antibody complex was recovered with 30 μl of protein A Sepharose beads (Sigma-Aldrich) after 3 hours with agitation at 4°C, and then the beads were washed twice in lysis buffer, washed once in PTEN reaction buffer [25 mM tris-HCl (pH 7.4), 140 mM NaCl, 2.7 mM KCl, and 10 mM DTT], and resuspended in 80 μl of PTEN reaction buffer. PTEN phosphatase activity was measured using the Malachite Green Assay Kit (Echelon Biosciences) and PIP3 as substrate according to the manufacturer’s instructions.
4
Phosphatase Activity Assay of PTEN Proteins
5
Purification and In Vitro Assay of INPP5K
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Wild-type and mutant GST-tagged full-length INPP5K was expressed in BL21 pLysS cells and purified on GSA beads (Thermo Fisher Scientific) in assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM MgCl2) plus 1% Triton X-100 and EDTA-free protease inhibitors (Roche Diagnostics). After washing, aliquots of beads were run on Coomassie gels to determine the abundance of full-length fusion proteins. Beads bearing equal amounts of fusion proteins were incubated in assay buffer containing 135 μM PtdIns(4,5)P2diC8, and free phosphate was measured using the Malachite Green assay kit (Echelon Biosciences). Results of three independent experiments were presented as mean ± standard deviation. To minimize variability between purifications, all constructs were freshly prepared and purified in parallel for each experiment, and beads used in the assay were afterward run on Coomassie gels to confirm equal protein loading.
6
Phosphatase Activity Assay of MTM-1
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The phosphatase activity of MTM-1 was assayed using a Malachite green assay kit (Echelon Biosciences, Inc.) by measuring the liberated free phosphate after the phosphatase reaction. Experiments were performed according to the manufacturer’s instructions with some modifications. In brief, 500 ng His-tagged MTM-1 or MTM-1(C378S) was incubated with 1,000 pmol of substrate (DiC8 PtdIns3P, DiC8 PtdIns(3,5)P2, DiC8 PtdIns(4,5)P2, DiC8 PtdIns(3,4)P2, and DiC8 PtdIns(3,4,5)P3; Echelon Biosciences, Inc.) in a 25-µl reaction containing 50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 2-mM CaCl2, 2-mM DTT, and 5% glycerol overnight at 20°C. The substrate-only controls were prepared in the same way to measure the background phosphate. Phosphate standards were prepared by diluting the phosphate standard solution provided in the kit according to the instructions. 100 µl Malachite green solution was added to each reaction, control, and phosphate standard for 20 min at room temperature, and absorbance was measured at 620 nm. Averages of triplicate samples were used to draw the phosphate standard curve and determine the free phosphate in each reaction and control.
7
INPP5E Enzymatic Activity Assay
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Activity assays were performed essentially as described (Bielas et al., 2009 (link)). Briefly, HEK293T cell lysates were obtained as above, and their protein levels measured with Pierce BCA Protein Assay kit (Thermofisher). EGFP-INPP5E or its mutants were then immunoprecipitated from 1 mg of cell lysate by overnight rotation at 4 °C with GFP-Trap_MA beads (Chromotek). Beads were then washed thrice in buffer containing 50 mM Tris-HCl pH = 7.5 and 150 mM NaCl buffer (no protease inhibitors or detergent), and twice more in activity buffer (50 mM Tris-HCl pH = 7.5, 150 mM NaCl, 3 mM MgCl2 and 0.1% octyl-β-D-glucopyranoside (Alfa Aesar)). For the activity assays, beads were incubated in activity buffer supplemented with 120 µM diC8-PtdIns(4,5)P2, from Echelon Biosciences. After incubating the enzyme reactions for 20 min at 37 °C, the supernatant was retrieved from the beads and its phosphate concentration measured at 620 nm using the Malachite Green Assay Kit (Echelon Biosciences). Beads were then processed for western blot as above.
8
Phosphatase Activity Assay of Mtm6
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The phosphatase activity of Mtm6 was determined using a Malachite green assay kit (Echelon Biosciences, Inc., Salt Lake City, UT, United States). Cells expressing Mtm6-GFP were starved without cAMP pulses for 3 h. Cells were washed with wash buffer (20 mM Hepes pH7.2 and 150 mM NaCl), resuspended in lysis buffer (20 mM Hepes pH7.2, 150 mM NaCl, 0.5% NP-40, 5% glycerol, 1 mM DTT, and protease inhibitor), and incubated on ice for 5 min. Lysates were centrifuged for 10 min at 4°C. The supernatant was incubated GFP trap beads (Smart-Lifesciences) for 1 h at 4°C. Beads were washed with wash buffer and reaction buffer (20 mM Hepes pH7.2, 150 mM NaCl, 2 mM DTT, 2 mM CaCl2, and 5% Glycerol). Beads containing 200 ng Mtm6-GFP were incubated with 3,000 pmol substrate in a 25 µl reaction for 30 min at 22°C. 20 μl supernatant was mixed with 80 μl Malachite Green solution at room temperature for 30 min. Free phosphate released was measured at 620 nm wavelength.
9
Quantifying Endogenous PP2A Activity
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