PARylated and MARylated PARP1 proteins were prepared as described [26 (link)] in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 50 mM NaCl, 0.2 mM DTT, 200 μM NAD+ (Trevigen) and 130 ng activated DNA (BPS Bioscience). Briefly, for the PAR hydrolysis activity assays, 70 nM PARP1 (PARP1-HSA; Trevigen) was auto-modified as described in [26 (link)]. After 20 min incubation, of PARP1 was passed three times through SpinTrap G-25 (GE Healthcare). PARylated PARP1 substrate was used in 10 μL reaction. For the MARylated PARP1, 1 μM of PARP1-E988Q was used as a substrate. Reactions were stopped by the addition of PARP inhibitor Olaparib (1 μM). The MgCl2 (Sigma) concentration was adjusted to 15 mM to allow full hydrolase activity. Automodified PARP1 was then incubated for indicated times at 30°C with hydrolytic enzymes in 10 μL reaction. Concentrations of hydrolytic enzymes used are as indicated in figures. Reactions were stopped by addition of Laemmli loading buffer, samples boiled at 90°C for 1.5 minutes and analysed by NuPAGE Novex Bis-Tris 4–12% Gel using MOPS buffer (Invitrogen). Radiolabelled experiments were visualized by autoradiography.
Mops buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
MOPS buffer is a commonly used buffer solution in molecular biology and biochemistry laboratories. It is a zwitterionic organic compound that maintains a stable pH range, typically between 6.5 and 7.9, making it suitable for various biological applications. The buffer helps maintain the optimal pH environment for various enzymatic reactions and biomolecular interactions.
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Lab products found in correlation
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (12 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (12 mentions)
Bis tris gel, by Thermo Fisher Scientific (11 mentions)
Lds sample buffer, by Thermo Fisher Scientific (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Odyssey scanner, by LI COR (5 mentions)
Pvdf membrane, by Thermo Fisher Scientific (5 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Odyssey blocking buffer, by LI COR (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Nupage gel, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp, by Bio-Rad (4 mentions)
Immobilon fl pvdf membrane, by Merck Group (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Reducing agent, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Lds loading dye, by Thermo Fisher Scientific (4 mentions)
158 protocols using mops buffer
1
Enzymatic Hydrolysis of PARylated and MARylated PARP1
2
Quantifying Active GDF3 Levels in Tissue
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Active GDF3 levels were determined in fresh tissue samples not part of the plasma screen (Additional file 1 : Table S3). Hippocampal samples were a random subset picked blindly from the same donors as the cortical samples. All tissues or cells were lysed in RIPA buffer and total protein concentrations were determined with a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). 10–20 μg of total protein was loaded for each sample into pre-cast 4–12 % bis-tris gels and run with MOPS buffer (Invitrogen, Carlsbad, CA). Gels were transferred onto PVDF membranes (Millipore, Billerica, MA). Antigen specific primary antibodies were incubated overnight at 4 °C and detected with species-specific horseradish-peroxidase labeled secondary antibodies. An ECL Western Blotting Detection kit (GE Healthcare, Cleveland, OH) was used to obtain a chemiluminescence signal, which was detected using Amersham Hyperfilm ECL (GE Healthcare). Band quantification was performed using ImageJ software (version 1.46; NIH, Bethesda, MD). Bands of interest were normalized to actin or neuron specific enolase for a loading control. For active GDF3 we used anti-GDF3 antibodies from Novus Biologicals (Littleton, CO; NBP1-96508).
3
Western Blot Protein Analysis Protocol
4
Western Blot Analysis of Cellular Proteins
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Treated or infected cells were washed twice with PBS. Whole cell lysates were collected in SDS-PAGE loading buffer containing 4 M Urea (Sigma-Aldrich, U0631) and 50 mM Dithiothreitol (DTT; Sigma-Aldrich, D0632). Proteins were resolved on NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen, NP0322BOX) in MES (Invitrogen; NP0002) or MOPS buffer (Invitrogen, NP0001) and transferred onto 0.2 μm nitrocellulose membrane (Amersham, 15249794) for 90 min at 30 volts in Novex transfer buffer (Invitrogen, NP0006-1) according to the manufacturer’s instructions. Membranes were blocked in PBS with 5% FBS (Block) for a minimum of 1 h at room temperature. Membranes were incubated in primary antibody diluted in Block for a minimum of 1 h, washed three times with PBST for 5 min each, then incubated in secondary antibody diluted in Block for 1 h. Following three 5 min washes in PBST, one 5 min wash in PBS, and one rinse in Milli-Q H2O, membranes were imaged on an Odyssey Infrared Imager (LiCor). The intensity of protein bands was quantified with Odyssey Image Studio Software.
5
Purification of PKM2 Protein Complexes
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TBL3 transiently expressing PKM2 construct were lysed in the following buffer: 20 mM Tris HCL pH8, 150 mM NaCl, 0.6% NP-40 and 2 mM EDTA. Protein complexes were then affinity-purified on anti-Flag antibody-conjugated agarose (Sigma, Ref: A2220) for bovine lysates. Elution was performed using Flag peptide. After five washes, immunopurified complexes were resolved on 4–12% SDS-PAGE bis-Tris acrylamide gradient gel in MOPS buffer (Invitrogen, Ref: NP 0322 BOX, NP0001-02, respectively).
6
Western Blot Analysis of P-gp and BCRP
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Tissue samples of the motor cortex and SC (control n = 6, sporadic [sALS] n = 6, and familial [fALS], n = 5) were homogenized in lysis buffer (20 mM TRIS-HCl [pH 8], 137 mM NaCl, 1 mM Na3VO4, 8% glycerol, 1% triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL typsin inhibitor). Next, they were centrifuged for 15 minutes at 18 000g. Protein content was determined using the Bio-Rad (Hercules, CA) DC protein assay kit. Per sample, 40 µg of proteins was loaded on a NuPAGE 4%–12% Bis-Tris Midi protein gels, 26-well (Thermo Fisher Scientific, Waltham, MA) and electrophoreses was performed for 2 hours at 30 mA. The temperature of the running buffer (MOPS buffer [Invitrogen, Carlsbad, CA] 1:20) was maintained below 10°C. Gels were dry-blotted on nitrocellulose and these were probed for P-gp with MDR1/ABCB1(E1Y7S) rabbit mAb #13978 1:1000 (Cell Signaling Technologies, Leiden, the Netherlands) or for BCRP with BXP-53 1:400 (ab24115; Abcam). Secondary HRP-antirabbit or HRP antirat 1:2000 (both from DAKO, Santa Clara, CA), respectively, were used and the blot exposed to ECL reagent (35 µg/mL coumaric acid, 220 µg/mL luminol, 0.0075% H2O2 in 100 mM TRIS pH 8.5) and images were taken on a Chemidoc XRS+ (Bio-Rad) and analyzed using Image Lab software (v5.2.1).
7
Quantifying Colicins and Immunity Proteins
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Radioactive 14C‐Leu was used in the CFPS reaction at a final concentration of 10 μM. Total and soluble fractions of each reaction were heat‐denatured and reduced by dithiothreitol and electrophoresed on 4%–12% NuPAGE sodium dodecyl sulfate‐polyacrylamide gels in MOPS buffer (Invitrogen). The gels were stained with InstantBlue Coomassie stain (Expedeon) for 1 h, destained overnight in dH2O, soaked in Gel Drying Solution (Bio‐Rad) for 30 min, fixed with cellophane films, dried overnight in a GelAir Dryer (Bio‐Rad), and exposed for 72 h on Storage Phosphor Screens (GE Healthcare Biosciences). The autoradiograms were scanned with a Typhoon FLA7000 Imager (GE Healthcare Biosciences). To determine the proportion of colicins to immunity proteins, we followed Davarinejad's protocol in the ImageJ software.22 Briefly, the band intensity determined by ImageJ was used to calculate the mass fraction. The molar fraction was calculated as the mass fraction and molar mass. The average molecular weight and average number of leucines were calculated based on the molar fraction of each protein (Table S1 ). These average values were used separately to quantify colicins and immunity proteins in each radioactive sample measured with 14C‐leucine radioactive scintillation counting (Table 2 ).
8
SDS-PAGE Analysis of Lysed CLL Cells
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SDS-PAGE was performed on 3 × 106 lysed CLL cells and run with equal protein loading on 10% Nu-PAGE Bis-Tris gel (Invitrogen, Waltham, MA, USA) with MOPS buffer (Invitrogen, Waltham, MA, USA). Protein was quantified using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc, Hercules, CA, USA.) and the blots stained with the following primary antibodies; rabbit anti-GAB (Cell Signaling Technology Europe B.V., Leiden, Netherlands, Cat no. 3232S) and mouse anti-Hsc70 (Insight Biotechnology Ltd., Wembley, UK, Cat no. sc-7298). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit/anti-mouse (Agilent Technologies LDA UK Limited, Cheshire, UK, Cat nos. P0448/P0447). Images were captured using the ChemiDoc-It Imaging System and quantified using ImageJ (RRID:SCR_003070). Representative immunoblots are shown in Supplementary Fig. 3 (Fig. S4 ).
9
Intestinal SGLT1 Protein Detection
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Intestinal mucosa was scraped and homogenized in buffer containing protease inhibitor cocktail (250 mM sucrose, 10 mM triethanolamine, Sigma-Aldrich, St. Louis, MO and Roche Applied Science, Indianapolis, IN, respectively) and Halt™ phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA).26 (link), 27 (link) The homogenate was centrifuged at 4,000 g for 15 min. Pellets were resuspended and used for Western blotting. Equal lane loading (20 µg) was achieved using a detergent compatible protein assay (Bio-Rad, Richmond, CA). Samples were resolved on 4–12% NuPAGE gels in MOPS buffer (Invitrogen, Carlsbad, CA). Gel proteins were transferred to nitrocellulose membranes and incubated over night at 4°C with rabbit-raised primary antibody against SGLT1 (1:2,000).3 (link) Horseradish peroxidase enzyme activity was detected via enhanced chemiluminescence (Amersham, Piscataway, NJ). For verification of equal protein loading, the membrane was stripped (0.2 mol/l NaOH for 5 min) and reprobed with monoclonal anti-β-actin antibody (1:30 000; A2228, Sigma-Aldrich). Densitometric analysis was performed by ImageJ Software v1.48 (National Institutes of Health, Bethesda, MD).
10
Intestinal SGLT1 Protein Detection
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