Intracellular lactate levels were evaluated using a quantitative assay kit (Sigma-Aldrich, Madrid, Spain) following the manufacturer’s instructions. Briefly, cells were treated with AZD3965 (10–100 nM) or the vehicle (DMSO) with or without glucose overload (30 mM) under normoxic or hypoxic (1% O2) conditions for 48 h. Cells were then collected and lysed using the lysis buffer included in the kit. Then, lysates were filtered with a 10-kDa MWCO spin filter (Sigma-Aldrich, Madrid, Spain) to remove lactate dehydrogenase. Absorbance was quantified with the Synergy HT plate reader at 570 nm. Each sample was evaluated in duplicate and the experiment was repeated at least three times. The results expressed as nmol lactate/mg protein were normalized according to the protein concentration in each sample, which was quantified in cell lysates employing the BCA1 kit (Sigma-Aldrich, Madrid, Spain).
10 kda mwco spin filter
Manufactured by Merck Group
Sourced in Spain
The 10 kDa MWCO spin filter is a laboratory instrument used for molecular separation and purification. It is designed to retain molecules with a molecular weight greater than 10 kilodaltons (kDa) while allowing smaller molecules to pass through. The core function of this spin filter is to facilitate the concentration and desalting of protein samples during sample preparation for various analytical techniques.
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6 protocols using 10 kda mwco spin filter
1
Intracellular Lactate Quantification
2
Kinetic Assay for Rv2780 Protein
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The reaction buffer was 125 mM glycine/KOH (pH 10.2), increasing concentration of L-alanine (0, 10, 100 mM), 1.25 mM NAD+ and 6.026 pM of Rv2780 protein in a final volume of 200 μL. The reactions were carried out in 96-well plate at 37 °C. Inhibitors at indicated concentrated were incubated with Rv2780 protein before the reaction. The reaction was measured by the production of NADH via NAD+/NADH Assay Kit with WST-8 (Beyotime). For direct detection of enzymatic product pyruvate, the reaction mixture was deproteinized with a 10 kDa MWCO spin filter (Sigma-Aldrich Co. LLC.) before quantification of pyruvate level with Pyruvate Assay Kit (Abcam, ab65342).
3
Cryo-EM sample preparation protocol
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Purified Pp GS was exchanged into Buffer B using a 10 kDa MWCO spin filter (Millipore) and concentrated to 0.9 mg/mL. For grid preparation, Quantifoil R1.2/1.3 Cu 300 (Quantifoil) holey carbon grids were cleaned for 100 s using a PELCO easiGlow glow discharge cleaning system and 3 μL of the sample were applied at 95% humidity and 22 °C. Following a 10 s incubation period, the grids were blotted for 1.5 s and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica Microsystems).
4
Cryo-EM Sample Preparation for Lm GS
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Purified Lm GS was exchanged into Buffer B using a 10 kDa MWCO spin filter (Millipore) and concentrated to 0.9 mg/mL. For grid preparation, Quantifoil R1.2/1.3 Cu 300 (Quantifoil) holey carbon grids were cleaned for 100 s using a PELCO easiGlow glow discharge cleaning system and 3 μL of the sample were applied at 95% humidity and 22 °C. Following a 10 s incubation period, the grids were blotted for 1.5 s and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica Microsystems).
5
Determining NADPH/NADP+ Ratio and Glycolytic Metabolites
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The NADPH/NADP+ ratio was determined using an EnzyFluo™ NADP/NADPH Assay Kit (BioAssay) according to the manufacturer’s protocol. Cellular metabolites were extracted and spectrophotometrically detected as previously described [15 (link), 70 (link), 71 (link)]. To measure the intracellular 2- and 3-PG levels, cells were collected after incubation with CLip-PC@Blank-LC NPs, DTX, siPGAM1, CLip-PC@LC-DTX NPs, CLip-PC@LC-siPGAM1 NPs, CLip-PC@CO-LC NPs, or CLip-PC@CO-LC NPs (MMP-9) for 24 h. Each group of cells was homogenized in 1.5 mL of hypotonic lysis buffer (20 mM HEPES [pH 7.0], 5 mM KCl, 1 mM MgCl2, 5 mM DTT, and protease inhibitor cocktail). Subsequently, the homogenates were centrifuged at 4 °C for 10 min at 13,000 rpm, and the supernatants were applied to a 10 kDa MWCO spin filter (Millipore). The flow-through containing the metabolites was used for the measurements.
6
Sample Preparation for Cryo-EM
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Purified Sa GS was exchanged into Buffer B using a 10 kDa MWCO spin filter (Millipore) and concentrated to 1.0 mg/mL. For grid preparation, UltrAufoil R1.2/1.3 Cu 300 (Quantifoil) holey gold grids were cleaned for 180 s using a PELCO easiGlow glow discharge cleaning system and 3 μL of the sample were applied at 95% humidity and 22 °C. Following a 10 s incubation period, the grids were blotted for 1.5 s and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica Microsystems).
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