Organic solvents for chromatography, MS grade water and MS grade formic acid were obtained from VWR (Darmstadt, Germany). Organic solvents used for preparative, semi-preparative and analytical chromatography were of gradient grade quality and water was bi-distilled water. Solvents used for LC-MS analyses were of MS grade quality. Formic acid used for chromatography was of MS grade quality. NADPH was obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). DL-glyceraldehyde and farnesal were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). A standardised solution of iso-α-acids produced from CO2 hop extract (30% w/w) was obtained from Barth Haas UK Limited (Tonbridge, UK). Mixtures of α-acids were kindly provided by Dr. Martin Biendl (Hopsteiner—HHV GmbH, Mainburg, Germany).
Nadph
Manufactured by Carl Roth
Sourced in Germany
NADPH is a coenzyme that plays a crucial role in numerous biochemical reactions within living organisms. It serves as a reducing agent, providing electrons and hydrogen atoms for various enzymatic processes. NADPH is an essential cofactor in many metabolic pathways, including biosynthesis, energy production, and cellular defense mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione reductase, by Merck Group (4 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Farnesal, by Merck Group (2 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (2 mentions)
Ms grade water, by Avantor (1 mentions)
Ms grade formic acid, by Avantor (1 mentions)
Dl glyceraldehyde, by Merck Group (1 mentions)
L xylose, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Roti quant, by Carl Roth (1 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Trypsin v5111, by Promega (1 mentions)
Nadph, by ITW Reagents (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Carl Roth (1 mentions)
Hepes, by Merck Group (1 mentions)
Crotonic anhydride, by Merck Group (1 mentions)
Coenzyme a, by Roche (1 mentions)
Du800, by Beckman Coulter (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Coomassie brilliant blue g250, by Carl Roth (1 mentions)
15 protocols using nadph
1
Organic Solvents for Chromatography
2
Carbohydrate and Cofactor Sourcing
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NADH, as disodium salt hydrate, L-xylose and Bradford reagent were ordered from Sigma-Aldrich (St. Louis, MO, USA); d -lyxose from Carbosynth (San Diego, CA, USA); D-arabinose from TCI Europe (Antwerp, Belgium); NADPH, as tetrasodium salt, and all other chemicals were ordered from Carl Roth (Karlsruhe, Germany).
3
Glutathione Quantification Assay
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Cells were lysed by 1 % sulfosalicylic acid (100 μl/well of 96 well plates). Cell lysate (20 μl) was diluted by the addition of 80 μl water. The reaction was initiated by the addition of 100 μl reaction buffer (100 mM sodium phosphate, pH 7.4), containing 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB; 100 μM, Sigma), NADPH (200 μM, Roth), glutathione reductase (1 U/ml, Sigma), and EDTA (1 mM, Sigma). For normalization, total protein content was detected (ROTIQuant, Roth). If not otherwise indicated, the detection of glutathione included both reduced (GSH) and oxidized (GSSG) glutathione.
4
Optimization of Thiol-Based Redox Assays
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HPDP-biotin (21341) and TCEP solution (77720) were purchased from Thermo Scientific. Soft release avidin resin (V2012) and trypsin (V5111) were obtained from Promega. NDA (70215) was ordered from Sigma-Aldrich and mBBr (596105) from Merck Millipore. GSSG was obtained from Serva and NADPH from Roth. All other reagents and the solvents were purchased from Sigma-Aldrich. T(SH)2 and Gsp were enzymatically prepared and the disulfide form was generated by reaction with a stoichiometric concentration of H2O2 (10 (link), 38 (link)). Recombinant T. brucei Px III was prepared as described (15 (link)). Human GR was a kind gift from Dr. Heiner Schirmer, Heidelberg University. Human Grx1 was purchased from Biomol.
Polyclonal rabbit antibodies against T. brucei Prx (15 (link)) and TryS (11 (link)) were generated previously. Rabbit antibodies against T. brucei aldolase were kindly provided by Dr. Christine Clayton, Heidelberg University, and the monoclonal mouse anti-β-tubulin antibodies were from Dr. Keith Gull, University of Oxford. Rabbit anti-Prx-SO3 antibodies were purchased from Abcam. HRP-conjugated goat antibodies against rabbit and mouse IgGs were from Santa Cruz Biotechnology. The Super Signal West Pico Kit was from Thermo Scientific.
Polyclonal rabbit antibodies against T. brucei Prx (15 (link)) and TryS (11 (link)) were generated previously. Rabbit antibodies against T. brucei aldolase were kindly provided by Dr. Christine Clayton, Heidelberg University, and the monoclonal mouse anti-β-tubulin antibodies were from Dr. Keith Gull, University of Oxford. Rabbit anti-Prx-SO3 antibodies were purchased from Abcam. HRP-conjugated goat antibodies against rabbit and mouse IgGs were from Santa Cruz Biotechnology. The Super Signal West Pico Kit was from Thermo Scientific.
5
Quantification of Total Glutathione
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For the detection of total glutathione (GSH and GSSG), samples (30 μl) were transferred to a 96-well plate and supplemented with 70 μl H2O. The recycling reaction was initiated by the addition of reaction buffer (sodium phosphate, 100 mM, pH 7.4), containing EDTA (1 mM), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (100 μM) (Sigma), NADPH (100 μM) (Carl Roth), and glutathione reductase (1 U/ml; from baker’s yeast) (Sigma). A standard curve with GSH (from 0 μM to 10 μM) was prepared in parallel. After 5 min, DTNB-thiol interaction was analyzed at 405 nm. For the detection of GSSG, a share of the samples was treated first with 2-vinylpyridine in a 20:1 (sample: 2-VP) stoichiometry for 1 h at RT for scavenging of reduced GSH. A GSSG standard curve was prepared (from 0 μM to 10 μM), the samples were then analyzed as described above. To obtain GSH values, oxidized glutathione (GSSG) results were subtracted from total glutathione (GSH + GSSG) values.
6
Glutathione Reductase Activity Assay
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The method is based on the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) by GR reducing oxidized L-glutathione (PanReac Applichem, Darmstadt, Germany) [107 (link),108 (link),110 (link)]. The buffer consisted of 50 mM (pH 8.00) HEPES (Sigma Aldrich, St. Louis, MO, USA), 20 mM EDTA and 5 mM NADPH (CarlRoth, Karlsruhe, Germany). The filtered extract was mixed with buffer and substrate, and the analyses were conducted at a wavelength of 340 nm. The kinetic alteration in GR (downward trajectory) was observed at intervals of 35 s. Based on the total protein concentration in the tissue, GR activity was converted to its activity in the fresh leaf biomass (µmol/mg):
where Sc is the slope coefficient (the highest value of absorption-the lowest value of absorption)/time of reaction (minutes); Vt is the total sample volume; Ve is the total extract volume; and Peq is the BSA equivalent based on standard curve.
where Sc is the slope coefficient (the highest value of absorption-the lowest value of absorption)/time of reaction (minutes); Vt is the total sample volume; Ve is the total extract volume; and Peq is the BSA equivalent based on standard curve.
7
Enzymatic Synthesis of Crotonyl-CoA
8
Measuring Cellular Glutathione Levels
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Cells were cultivated as described and counted in a Neubauer chamber and adjusted to the same cell number (2 × 107). After washing in phosphate buffer (80 mM) containing 3.5 mM EDTA, cells were lysed by vortexing with glass beads and centrifuged at 15,800×g for 1 min. The supernatant was used to measure the GSH content according to Griffith [29] (link). The reaction mixture contained 12.5 µL supernatant, 5 µL GR (20 units mL−1; Sigma-Aldrich, Steinheim, Germany), 50 µL of 6 mM DTNB (Ellman's reagent, Sigma-Aldrich, Steinheim, Germany), and 350 µL of 0.3 mM NADPH (Carl Roth, Karlsruhe, Germany) in phosphate buffer. Distilled water was added to a final volume of 750 µL (final phosphate buffer 80 mM, 3.5 mM EDTA). The increase of optical density at 412 nm was measured in intervals of 30 s over 10 min using a spectrophotometer (DU800, Beckman Coulter GmbH, Krefeld, Germany).
9
Biochemical Assay Protocol for ATP
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10
Redox Regulation of CP12 Proteins
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CrNTRC, OsNTRC, CrCP12, and AtCP12 recombinant proteins used in this assay were prepared as described in SI Appendix, Text , while reaction buffer used was IP lysis buffer. Then, 100 µg CP12 proteins were mixed with 150 µg NTRC proteins, 30 mM NADPH (Carl Roth, #AE14) or both. For untreated, positive, and negative controls, 100 µg CP12 or PRX1 proteins were treated with blank buffer, 5 mM reduced DTT (AppliChem, #A2948) or oxidized DTT (Sigma Aldrich, #D3511), respectively. All reactions were equalized to a final volume of 20 µL in PCR tubes and incubated at 37 °C for 1.5 h. Subsequently, each reaction was stopped by adding 7 µL ROTI®Load2 (4×) nonreducing loading dye (Carl Roth, #K930). The proteins were then separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250 (Carl Roth #9598).
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