An IEF gel (Novex pH 3-10 IEF Protein Gels, Thermo Fisher Scientific) was set in an electrophoresis device (XCell SureLock Mini-Cell, Thermo Fisher Scientific), and IEF running buffers (Novex IEF pH 3-10 Cathode Buffer and Novex IEF Anode Buffer, Thermo Fisher Scientific) were loaded. The samples, including CRP, labeled CRP, anti-CRP mAb, and BSA, as well as the IEF marker (IEF marker 3-10, Thermo Fisher Scientific) were diluted with a sample buffer (Novex IEF Sample Buffer pH 3-10, Thermo Fisher Scientific) and loaded onto the gel. IEF was performed under the following sequential conditions: (i) 100 V constant for 1 h, (ii) 200 V constant for 1 h, and (iii) 500 V constant for 0.5 h. After the run, the samples were fixed for 1 h with a 12% trichloroacetic acid solution (Hayashi Pure Chemical). Subsequently, the gel was rinsed with deionized water and stained for 1 h with Coomassie Brilliant Blue (EzStain AQua, ATTO) and finally destained with deionized water.
Novex ph 3 10 ief protein gels
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Novex™ pH 3-10 IEF Protein Gels are a lab equipment product designed for isoelectric focusing (IEF) of proteins. They provide a pH gradient range of 3-10 to facilitate the separation and analysis of proteins based on their isoelectric points.
Automatically generated - may contain errors
Lab products found in correlation
Api electrospray single quadrupole msd, by Agilent Technologies (2 mentions)
Ief marker 3 10, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Precision plus protein unstained standard, by Bio-Rad (1 mentions)
Mini protein tetra system, by Bio-Rad (1 mentions)
Marker proteins, by GE Healthcare (1 mentions)
Nativepage novex bis tris gel system, by Thermo Fisher Scientific (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
6 protocols using novex ph 3 10 ief protein gels
1
Isoelectric Focusing of Proteins
2
Characterization of Peptide PEP40233
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Characterization of PEP40233 was performed to determine purity, isoelectric point (pI) and thermal stability, prior to performing biodistribution studies. The lyophilized molecule was dissolved in Dulbecco’s phosphate-buffered saline (DPBS) to a concentration of 1 mg/mL. To determine the purity, a sample of PEP40233 was loaded on an RP-UPLC column (Agilent Zorbax 300 SB-C8 RRHD; 1.8 µm, 2.1 × 100 mm column) and eluted by a linear gradient of acetonitrile from 25–50% in 0.1% TFA during 8.3 min at a flow rate of 0.5 mL/min, at 40 ℃. The molecular mass of the construct was determined using mass-spectrometry (API electrospray single quadrupole MSD, Agilent Technologies, Santa Clara, CA, USA). Isoelectric focusing was applied to determine the pI of PEP40233 (Novex™ pH 3-10 IEF Protein Gels, ThermoFisher, Manassas, MA, USA). The reversible folding ability of PEP40233 after heating to 90 ℃ was analyzed by circular dichroism spectroscopy (Jasco J-810 spectropolarimeter, Jasco Scandinavia AB, Mölndal, Sweden).
3
Protein Extraction and Analysis
4
Laccase Characterization by SDS-PAGE and Native-PAGE
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE under non-denaturing conditions (native-PAGE) was conducted with a 10% Mini-PROTEAN TGX precast gel using Precision Plus Protein Unstained Standards (Bio-Rad, California, USA) in a Mini Protein Tetra system (Bio-Rad, California, USA) according to the manufacturer’s instructions. SDS-PAGE protein bands were visualized using Coomassie brilliant blue staining, whereas the native-PAGE enzyme band was visualized by incubating the gel in 1 mM guaiacol in 100 mM sodium acetate buffer (pH 4.0) at room temperature for 20 min. The isoelectric point (pI) of laccase was determined using the XCell SureLock Mini-Cell electrophoresis system (Thermo Fischer Scientific, Massachusetts, USA) with Novex pH 3–10 IEF Protein Gels (Thermo Fisher Scientific, USA) and the IEF Standards with a pI 4.45–9.6 (BioRad, Hercules, USA) according to manufacturer’s instructions.
5
Protein Characterization using PAGE
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The proteins were routinely analyzed on 15% polyacrylamide gels under denaturing and reducing conditions, and visualized using Coomassie brilliant blue staining or silver staining. Low molecular weight markers of 14.4 kDa to 97 kDa (GE Healthcare Life Sciences, Buckinghamshire, England) were used for molecular mass estimations. The proteins were analyzed under non-denaturing conditions using blue native PAGE with a Novex NativePAGE Bis-Tris gel system with 4% to 16% gradient protein gels (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. NativeMark unstained protein standards (ThermoFisher Scientific) was used for the molecular mass estimations. Isoelectric focusing was carried in precast Novex pH 3–10 IEF protein gels (ThermoFisher Scientific) following the manufacturer instructions. Marker proteins with pI values from 3.5 to 9.3 were used for calibration (GE Healthcare).
6
Biophysical Characterization of Antibody Constructs
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The characterization of ABY-027 and ABY-271 was performed to determine thermal stability, purity and isoelectric point (ABY-271) before performing biodistribution studies. The lyophilized molecules were dissolved in PBS containing 0.5 mM EDTA to a concentration of 1 mg/mL.
The molecules were characterized by Circular Dichroism (Jasco J-810 spectropolarimeter, Jasco Scandinavia AB) for reversibility of structure after heating to 90 °C, and with RP-UPLC (Agilent Zorbax 300 SB-C8 RRHD; 1.8 µm, 2.1 × 100 mm column). Elution by linear gradient of acetonitrile from 25–50% in 0.1% TFA during 8.3 min at a flow rate of 0.5 mL/min and at 40 °C. The correct molecular mass of the construct was confirmed using mass-spectrometry (API electrospray single quadrupole MSD, Agilent Technologies, Santa Clara, CA, USA). Isoelectric focusing was used to determine the isoelectric point for ABY-271 (Novex™ pH 3-10 IEF Protein Gels, ThermoFisher, Manassas, MA, USA).
The molecules were characterized by Circular Dichroism (Jasco J-810 spectropolarimeter, Jasco Scandinavia AB) for reversibility of structure after heating to 90 °C, and with RP-UPLC (Agilent Zorbax 300 SB-C8 RRHD; 1.8 µm, 2.1 × 100 mm column). Elution by linear gradient of acetonitrile from 25–50% in 0.1% TFA during 8.3 min at a flow rate of 0.5 mL/min and at 40 °C. The correct molecular mass of the construct was confirmed using mass-spectrometry (API electrospray single quadrupole MSD, Agilent Technologies, Santa Clara, CA, USA). Isoelectric focusing was used to determine the isoelectric point for ABY-271 (Novex™ pH 3-10 IEF Protein Gels, ThermoFisher, Manassas, MA, USA).
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