To generate the Fab from the IgG, a stop codon was inserted in the heavy chain constant region at “KSCDK”. The truncated heavy chains were co-transfected with the corresponding light chains in 293Expi cells to produce the Fabs. The supernatants were harvested 4 days post transfection. Fabs were purified with CaptureSelect™ CH1-XL MiniChrom Columns (#5943462005). Supernatants were loaded onto columns using an Econo Gradient Pump (Bio-Rad #7319001). Following a wash with 1x PBS, Fabs were eluted with 25 mL of 50 mM acetate (pH 4.0) and neutralized with 2 M Tris Base. The eluate was buffer exchanged with 1x PBS in 10K Amicon tubes (Millipore, UFC901008) and filtered with a 0.22 μm spin filter.
Econo gradient pump
Manufactured by Bio-Rad
Sourced in United States
The Econo Gradient Pump is a laboratory equipment designed for the automated creation and delivery of linear gradients. It is capable of precisely mixing and delivering two different liquid solutions at variable flow rates to generate a continuously changing concentration profile, commonly used in chromatographic separations and other analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
10k amicon tubes, by Merck Group (4 mentions)
Econo uv monitor, by Bio-Rad (2 mentions)
Gradient master, by BioComp Instruments (2 mentions)
Deuterium oxide, by Cambridge Isotopes (1 mentions)
1 2 13c acetate, by Cambridge Isotopes (1 mentions)
1 13c glucose, by Merck Group (1 mentions)
1 13c glucose, by Bio-Rad (1 mentions)
1 2 13c acetate, by Bio-Rad (1 mentions)
Rodent tail vein catheter and restraining apparatus, by Braintree Scientific (1 mentions)
Hrp labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Optima l 70k, by Beckman Coulter (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
Rnasin plus rnase inhibitor, by Promega (1 mentions)
Model 2110 fraction collector, by Bio-Rad (1 mentions)
Hitrap column, by Cytiva (1 mentions)
Expi293f, by Thermo Fisher Scientific (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
14 protocols using econo gradient pump
1
Fab Generation from IgG
2
Isotope-Labeled Glucose and Acetate Metabolism
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R-sodium lipoic acid was a gift from GeroNova Research. A constant infusion of [1-13C]-glucose and [1,2-13C]-acetate was performed by using the ECONO gradient pump (Bio-Rad Laboratories, Hercules, CA, USA) and Rodent tail vein catheter and restraining apparatus (Braintree Scientific). Deuterium oxide (99.9%) and [1,2-13C]-acetate (99%) (Cambridge Isotope Laboratories, Andover, MA, USA), [1-13C]-glucose (99%) (Sigma-Aldrich, St Louis, MO, USA) were used for the NMR experiments. Chemicals of the purest grade were used for all assays. The primary antibodies against β-actin (SC-1616), GLUT3 (SC-74399), GLUT4 (sc-1608), Na+/K+-ATPase (SC-58628), and HRP-labeled secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies for Akt (9272) and p-Akt (Ser473) (9271) were purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Forizyme Production and Purification
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All forizymes were produced in 200-ml yeast cultures and were purified as previously described24 (link) with the following modifications. Briefly, yeast cells were grown to an OD of 5 and digested with 300 U of Arthrobacter luteus lyticase following a washing step in Ca2+-free Tris buffer (see above) plus a cOmplete Mini protease inhibitor tablet. After mechanical cell disruption for 12 min as described above, the cells were loaded onto a 72.8–105.2% (w/v) Nycodenz® (Medinor AS, Norway) density gradient (10 ml volume) prepared with a gradient mixer in combination with the Econo Gradient Pump (Bio Rad, Germany) and centrifuged for 2.5 h at 4 °C and 247,103.6 x g in an Optima L-70 K ultracentrifuge (Beckmann Coulter, Germany). The band containing the forizymes was isolated, washed with Ca2+-free Tris buffer (see above), and analysed by microscopy to determine the purity. If necessary, purification was improved by loading the isolated forizymes onto a second 72.8–105.2% (w/v) Nycodenz® density gradient (5 ml volume). After centrifugation for 1.5 h at 4 °C and 245,418.9 x g, the forizyme band was isolated and resuspended in 800 μl Ca2+-free Tris buffer (see above). The production and purification was carried out three times using independent batches, and all subsequent experiments were performed independently for each batch unless indicated otherwise.
4
Purification of Eukaryotic 80S Ribosomes
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Gradient preparation: SG 50 Gradient Maker (GE Healthcare) is used to make a linear gradient of 15–30%, wherein the higher % sucrose solution is loaded in the mixing chamber and the lower % sucrose solution is loaded in the other, allowing to mix slowly. The outlet is connected with a pump and sucrose is collected drop wise from the outlet.
The supernatant is treated with 1 mM puromycin for 30 min at 4°C (34 (link)) with intermittent mixing and loaded on 15–30% sucrose gradient prepared in Buffer A. The samples are centrifuged at 25 000 rpm for 11 h in a SW-28 rotor and fractions are collected from bottom to top using an Econo Gradient Pump (Biorad) with an Econo UV Monitor (Biorad) and a Fraction Collector. The sample absorbance is recorded using UV reader (Biorad) and the peak corresponding to 80S is pooled for PEG20K precipitation (7 (link)). A final concentration of 7% PEG20K is added to the pooled fractions, incubated on ice for 10 min and centrifuged at 17 400g for 10 min. The pure ribosomal pellet is dissolved in resuspension buffer C and filtered using 0.22 µm filters (Millipore) for further analysis or stored without filtration on ice for 7 days for crystallization. Snap freezing and storage is not advised. For concentration calculations, 1 A260 unit corresponds to 20 pmol of 80S ribosome.
The supernatant is treated with 1 mM puromycin for 30 min at 4°C (34 (link)) with intermittent mixing and loaded on 15–30% sucrose gradient prepared in Buffer A. The samples are centrifuged at 25 000 rpm for 11 h in a SW-28 rotor and fractions are collected from bottom to top using an Econo Gradient Pump (Biorad) with an Econo UV Monitor (Biorad) and a Fraction Collector. The sample absorbance is recorded using UV reader (Biorad) and the peak corresponding to 80S is pooled for PEG20K precipitation (7 (link)). A final concentration of 7% PEG20K is added to the pooled fractions, incubated on ice for 10 min and centrifuged at 17 400g for 10 min. The pure ribosomal pellet is dissolved in resuspension buffer C and filtered using 0.22 µm filters (Millipore) for further analysis or stored without filtration on ice for 7 days for crystallization. Snap freezing and storage is not advised. For concentration calculations, 1 A260 unit corresponds to 20 pmol of 80S ribosome.
5
Ribosomal Subunit Purification Protocol
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Equipments required: 10-l flasks for cell culture, SW-28 rotor, Type 50.2 Ti Beckman-Coulter rotor, GE SG-50 Gradient maker, Econo UV Monitor (Biorad), a Fraction Collector (Biorad), Econo Gradient Pump (Biorad).
Deionized distilled water is used for buffer preparations, and complete protease inhibitor (Roche) is added to all the buffers. Also, sucrose solution must be treated with bentonite after preparation with buffer A to inhibit ribonucleases (31 (link),32 (link)) if present. Buffer A contains 20 mM Tris, pH 7.5, 2 mM Mg(OAc)2, 150 mM KCl. Buffer B contains 20 mM Tris, pH 7.5, 6 mM Mg(OAc)2, 150 mM KCl, 6.8% sucrose, 1 mM DTT, RNasin Plus RNase Inhibitor (Promega). Resuspension buffer C contains 100 mM KCl, 5 mM Mg(OAc)2, 20 mM HEPES, pH 7.6, 1 mM DTT, 10 mM NH4Cl. For 60S and 40S subunit purification a slightly modified buffer A is required containing 20 mM Tris, pH 7.5, 2 mM Mg(OAc)2, 500 mM KCl. The role of ion concentration in inter- and intra-subunit interaction is discussed in the ‘Results’ section.
Deionized distilled water is used for buffer preparations, and complete protease inhibitor (Roche) is added to all the buffers. Also, sucrose solution must be treated with bentonite after preparation with buffer A to inhibit ribonucleases (31 (link),32 (link)) if present. Buffer A contains 20 mM Tris, pH 7.5, 2 mM Mg(OAc)2, 150 mM KCl. Buffer B contains 20 mM Tris, pH 7.5, 6 mM Mg(OAc)2, 150 mM KCl, 6.8% sucrose, 1 mM DTT, RNasin Plus RNase Inhibitor (Promega). Resuspension buffer C contains 100 mM KCl, 5 mM Mg(OAc)2, 20 mM HEPES, pH 7.6, 1 mM DTT, 10 mM NH4Cl. For 60S and 40S subunit purification a slightly modified buffer A is required containing 20 mM Tris, pH 7.5, 2 mM Mg(OAc)2, 500 mM KCl. The role of ion concentration in inter- and intra-subunit interaction is discussed in the ‘Results’ section.
6
FPLC Fractionation of Toluene Synthase
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FPLC fractionation was performed in an anaerobic globe box. Crude cell-free extract supernatants were applied to a Bio-Scale Mini CHT-II ceramic hydroxyapatite column (5-mL bed volume, 40-μm particle diameter; Bio-Rad, Hercules, CA, USA) with a Bio-Rad Econo Gradient Pump. The chromatographic conditions were as follows: using a flow rate of 1 mL/min and a binary eluent system of 10 mM and 500 mM potassium phosphate buffer (pH 7.5; also containing 2 mM DTT), the column was held at an initial phosphate concentration of 10 mM for 1 column volume and protein was then eluted with a linear gradient from 10 to 500 mM phosphate at a rate of 49 mM/mL. One-mL fractions were collected with a model 2110 fraction collector (Bio-Rad) and assayed for phenylacetate decarboxylase activity (as described above). Attempts were made to further purify toluene synthase activity with a quaternary ammonium anion exchange column (Bio-Scale Mini UNOsphere Q) after buffer exchange of active fractions from the hydroxyapatite column, however, these attempts were unsuccessful and are not further reported here.
7
Laccase Purification and Characterization
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The culture supernatant was collected by centrifugation, filtrated, and concentrated using an Amicon ultrafiltration device Stirred Cell 8400 (membrane cutoff 10 kDa, Merck Millipore). The concentrate was dialyzed against 20 mM piperazine buffer pH 5.7 at 4 °C overnight, and applied to an equilibrated Q sepharose column with a flow of 4 mL min−1. A linear gradient from 0 to 500 mM NaCl was applied with a Biorad Econo gradient pump. Eluted fractions were analyzed for laccase activity, and selected positive fractions were pooled together, and dialyzed again. A second purification step followed, applying the pooled and dialyzed fractions to a properly equilibrated DEAE-Sepharose column. Elution was performed with a linear gradient from 0 to 500 mM NaCl as previously described. Fractions with laccase activity were pooled, concentrated, and dialyzed against 20 mM Bis–Tris buffer pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [53 (link)], with 12.5% polyacrylamide gels.
8
Purification of His-tagged Proteins
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An overnight culture was used to inoculate 1 liter of preheated LB containing appropriate antibiotics for maintenance of the overexpression plasmid to an OD600 of 0.05. At an OD600 of approximately 0.4, 1 mM IPTG was added, and the culture incubated for a further 4 h. Cells were harvested by centrifugation and stored as pellets at −80°C. Pellets were freeze-thawed three times in sodium phosphate buffer and sonicated on ice six times. Insoluble material was separated by centrifugation at 10,000 × g for 30 min. The supernatant was filter sterilized (0.45-µm filters) and purified using a 5-ml HiTrap column (Amersham) with a BioRad Econo gradient pump and fraction collector. The His-tagged proteins were eluted from the column using an isocratic gradient of 5-to-60% 0.5 M imidazole over 30 min. Eluted fractions were analyzed by SDS-PAGE. Fractions containing overexpressed protein were pooled, transferred to dialysis tubing, and dialyzed three times in phosphate-buffered saline (PBS) for 18 h in total. The identities of purified, overexpressed proteins were confirmed by N-terminal sequencing.
9
In Vitro Culture and Characterization of Engineered Vascular Constructs
10
Ribosome profiling of Trypanosoma cruzi
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At least 1 × 10 9 exponentially growing epimastigotes, before or after pre-incubation with TAU medium for 2 hr were treated with 100 μg/ml cycloheximide at 28°C for 10 min, followed by ice cooling for additional 10 min. The cells were then collected by centrifugation at 2,000 g for 5 min at 4°C, washed in ice-cold PBS containing the same concentration of cycloheximide and suspended in approximately 300 μl of ice-cold Buffer A (10 mM Tris-HCl, pH 7.4, 300 mM KCl, 10 mM MgCl 2 , 1 mM dithiothreitol and 100 μg/ml cycloheximide). A drop of Triton X-100 was added to the lateral side of tube to reach 1% (wt/vol) and the tubes were repeatedly mixed by inversion for about 3-5 min. The lysates were centrifuged at 6,000 g for 3 min at 4°C and the supernatants were transferred to a new tube. Stocks of heparin at 100 mg/ml and NaCl at 5 M were added to 1 mg/ml and 150 mM respectively. Samples containing the equivalent of 10 absorbance units at 260 nm were then loaded on the top of a 7-47% sucrose gradient in Buffer A, prepared using a Gradient Master (Biocomp). The tubes were centrifuged at 250,000 g for 2.5 hr in a Beckman SW41 rotor at 4°C. The gradient was collected from the top by injecting the bottom with a continuous flow of 60% sucrose at 1 ml/min using an Econo Gradient Pump (Bio-Rad) and the fractions were monitored by reading the absorbance at 254 nm.
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