CTPR2a and CTPR2n were purified from cell pellets of 500 mL cultures. Pellets were resuspended in lysis buffer (50 mM sodium phosphate pH 8.5, 150 mM NaCl, 30 mM imidazole, SIGMAFAST protease inhibitor cocktail EDTA-free (1 tablet/100 mL, Sigma), 400 U/mL DNase I (Sigma)) and lysed using an EmulsiFlex C5 homogenizer (Avestin) at 15000 psi. Cell debris was removed by centrifugation (40000 g, 30 min, 4 °C). Supernatant was filtered through a 0.22 μm syringe filter (Jet Biofil) and loaded onto a 5 mL HisTrap excel column (GE Healthcare). The column was washed with wash buffer (50 mM sodium phosphate pH 8.5, 150 mM NaCl, 30 mM imidazole) and protein was eluted with elution buffer (50 mM sodium phosphate pH 8.5, 150 mM NaCl, 300 mM imidazole). Elution fractions were analyzed on SDS-PAGE and pure fractions were pooled.
Sigmafast edta free protease inhibitor cocktail
Manufactured by Merck Group
The SigmaFast EDTA-free protease inhibitor cocktail is a ready-to-use solution designed to inhibit a broad spectrum of proteases. It is formulated without EDTA, making it suitable for applications where the presence of chelating agents may be undesirable.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
Emulsiflex c5 homogenizer, by Avestin (1 mentions)
Nativepage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Dtx 880 multimode plate reader, by Beckman Coulter (1 mentions)
Cyclohexamide, by Merck Group (1 mentions)
Sigmafast protease inhibitor cocktail, by Merck Group (1 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ipegal ca 630, by Merck Group (1 mentions)
Hei torque core, by Heidolph (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Immuno blot polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Protein g sepharose beads, by Thermo Fisher Scientific (1 mentions)
Cdp star western blot chemiluminescence reagent, by PerkinElmer (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
9 protocols using sigmafast edta free protease inhibitor cocktail
1
Purification of CTPR2a and CTPR2n Proteins
2
Analyzing Protein Complexes via Blue Native Gel
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Blue Native gel analysis was performed according to manufacturer instructions (Novex). Samples were prepared as follows: Mid-Log phase culture cells were washed twice in PBS, resuspended in 24 μL extraction buffer (25 mM HEPES, 150 mM sucrose, 20 mM potassium glutamate, 3 mM MgCl2, 0.5% NP40, 150 mM KCl, 0.5 mM DTT, and SIGMAFAST Protease Inhibitor Cocktail, EDTA-free [Sigma-Aldrich]), incubated on ice for 20 min, and centrifuged at full speed for 10 min/4°C. Eighteen microliters of the supernatant was added to 6.25 μL of 4× Native PAGE buffer and 1 μL of G-250 Coomassie sample buffer. The samples were electrophoresed through precast 4%–16% NativePAGE Novex Bis-Tris gels following the manufacturer specifications (Life Technologies). The NativeMark Unstained Protein standard (Life Technologies) was used to estimate complex sizes. Proteins were transferred to Immun-Blot PVDF 0.2 μm membranes (BioRad). Membranes were fixed in 8% acetic acid for 15 min, rinsed with water, and incubated with primary or secondary antibody. The size marker lane was removed prior to antibody incubation, air-dried, equilibrated with methanol, and stained with Coomassie dye for visualization.
3
Quantitative SpCas9 Protein Expression
4
Polysome Fractionation Workflow
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Stocks for polysome buffer (PSB; 50 mM Tris, pH = 7.5, 100 mM KCl, 12 mM MgCl2, 1% IPEGAL CA-630 [Sigma-Aldrich], plus: 1 mM DTT, 60 U/ml RiboLock RNase inhibitor [Thermo Fisher Scientific], 100 μg/ml Cyclohexamide [Sigma-Aldrich], 2× SigmaFast EDTA-free protease inhibitor cocktail [Sigma-Aldrich] dissolved in PSB stock), high salt wash buffer (HSB; 50 mM Tris pH = 7.5, 300 mM KCl, 12 mM MgCl2, 1% IPEGAL, plus: 1 mM DTT, 20 U/ml RiboLock, 100 μg/ml Cyclohexamide and 0.5× EDTA-free SigmaFast protease inhibitor cocktail), and extra high salt buffer (EHSB; HSB containing additional 300 mM NaCl) were prepared using RNase-free reagents and stored at 4°C. Inhibitors were added to stock solutions directly before use (indicated by “plus”). RNA purifications were performed in a Biosafety level 3 environment. Frozen tissue samples were homogenized at 450 rpm, using Wheaton Potter-Elvehjem homogenizers and PTFE pestles (DWK Life Science) with a motorized homogenizer (HEI-Torque Core, heidolph), in 200 μl ice-cold PSB per 0.01 g tissue. Homogenates were centrifuged at 4°C, 400g for 2 min to collect nuclei. Supernatant was transferred to fresh vials and centrifuged at 4°C, 10,000g for 10 min.
5
Protein Quantification and Immunoblotting Workflow
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Where described, whole cell lysates and mouse liver tissue samples were prepared using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 0.5 mM sodium orthovanadate, 1X SIGMAFAST EDTA free protease inhibitor cocktail [Sigma Aldrich, Saint Louis, MO]) with concentrations determined using the Pierce BCA Protein Assay (Thermo Scientific, Rockford, IL). Independent of the experiments performed, all samples were size-fractionated on 10% SDS-PAGE under reducing conditions and electro-transferred to immuno-blot polyvinylidene difluoride (PVDF) membrane (BioRad). Membranes were incubated with polyclonal anti-FETUB or fibrinogen γ antibodies (SantaCruz, Hercules, CA) and visualized with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz). Total protein in all experiments was assessed by Ponceau S staining and used for normalization (Sigma). Immunoreactive proteins were detected using enhanced chemiluminescence (Thermo Scientific, Pierce Biotechnology, Rockford, IL) with image capture performed using either a ChemiDoc Imager or ChemiDoc Touch Imaging System (BioRad). Results were quantified using QuantityOne or Image Lab Version 5.2.1 software, where the values are presented as arbitrary units (AU).
6
Immunoprecipitation and Western Blotting of Flt3 and Fes
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AML cells (2.5 x 106 in 5 mL) were cultured in the presence of kinase inhibitors or DMSO alone for 6 to 16 hours prior to lysis by sonication in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) supplemented with 1 mM sodium orthovanadate, 1 mM sodium fluoride, 5 units/ml Benzonase (Novagen), and SigmaFAST EDTA-free protease inhibitor cocktail (Sigma). Flt3 and Fes were immunoprecipitated from 1 mg of cell lysate with 2 μg of antibody and 25 μL of protein G-Sepharose beads (Life Technologies) overnight at 4°C. Immunoprecipitates were washed three times by resuspension in 1.0 ml of RIPA buffer. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with the indicated antibodies followed by alkaline phosphatase-linked secondary antibodies. CDP-Star Western Blot Chemiluminescence Reagent (Perkin-Elmer) was used for detection.
7
Preparation of Ribosome-Enriched Cell Extracts
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Vegetative cells were treated with 100 µg/mL cycloheximide for 5 min prior to harvesting. Cells were pelleted by centrifugation and resuspended in buffer KK2 (16.5 mM KH2PO4, 3.9 mM K2HPO4, 2 mM MgSO4) plus 100 µg/mL cycloheximide. They were washed twice more in KK2, with a final wash in KK2 containing 100 µg/mL cycloheximide and 1× SigmaFast EDTA-free protease inhibitor cocktail (Sigma #S8830). The cell pellet was resuspended at 2 × 108/mL in 50 mM HEPES pH 7.5, 40 mM Mg(CH3COO)2, 25 mM KCl, 5% sucrose, 0.4% IGEPAL® CA-630 (Sigma #I8896), 100 μg/mL cycloheximide, 1× SigmaFast EDTA-free protease inhibitor cocktail, 2 mM PMSF and lysed by passing through a 25 mm diameter Swin-Lok filter holder (GE Healthcare Life Sciences #420200) containing a prefilter (Millipore #AP1002500) together with a 5 µm nucleopore track-etched membrane (Whatman #110613). The lysate was cleared by centrifugation (8,000 g for 30 min at 4 °C) and the supernatant passed through a 33 mm Millex-® GV 0.22 µm PVDF filter unit (Millipore #SLGV033RS). The filtrate was divided into 1.4 mL aliquots after A260 determination, flash-frozen in liquid N2, and stored at −80 °C. All buffers were at 4 °C.
8
Western Blot Analysis of FGF21 Protein
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Protein samples were prepared by homogenising frozen tissue samples in RIPA buffer (Thermo Fisher®) supplemented with SIGMA FAST® EDTA‐free protease inhibitor cocktail (Sigma®). Protein concentrations were determined via the Pierce BCA Protein Assay Kit (Thermo Fisher®), and 20 μg of each sample was run on a 4%‐ to 20%‐gradient polyacrylamide gel (BioRad®). Standard western blotting protocols were followed utilising nitrocellulose membrane (BioRad®). Primary antibodies were obtained from Invitrogen® (Anti‐FGF21; MA 5–35418) and Cell Signaling® (Anti‐Beta Actin; 4970). The anti‐rabbit IgG DyLight™ 800 (Cell Signaling®; 5151) was used as the secondary antibody, and blots were scanned with the LiCor Odyssey® infrared scanner. The Odyssey software was utilised for detection and band intensity calculations. Signal intensities of FGF21 were determined relative to those of beta actin.
9
Yeast Cell Protein Extraction and Purification
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Yeast cells were grown at 30ºC in 200 ml of YPD medium to an OD 600 of around 0.8. Cells were washed in ice-cold dH 2 O and resuspended in 400 µl of lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1.5 mM MgCl 2 , 5% glycerol, 0.1% NP-40, 1 mM PMSF, and SIGMAFAST EDTA-free protease inhibitor cocktail (Sigma-Aldrich)). Cell extracts were obtained by glass bead lysis with a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) set at 5'000 rpm using a 3x 30 sec lysis cycle with 30 sec breaks in between at 4ºC. Cell lysates were clarified by centrifugation at 4ºC
for 10 min at 13'500 rpm. GFP-Trap Magnetic Agarose beads (Chromotek, Planegg-Martinsried,
for 10 min at 13'500 rpm. GFP-Trap Magnetic Agarose beads (Chromotek, Planegg-Martinsried,
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