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.
Ief marker 3 10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IEF Marker 3–10 is a ready-to-use solution containing a mixture of proteins with known isoelectric points (pI) ranging from 3 to 10. It is primarily used as a reference in isoelectric focusing (IEF) experiments to determine the pI of unknown proteins.
Automatically generated - may contain errors
3 protocols using ief marker 3 10
1
Isoelectric Focusing of Proteins
2
Isoelectric Focusing of Purified ML-II
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Purified ML-II was analyzed on Novex® IEF gels pH 3–7 (Invitrogen, Carlsbad, CA, USA) using a XCell SureLock®Mini-Cell, according to the manufacturer’s instructions. Briefly, 1× IEF anode and 1× cathode buffers were added to the lower and upper buffer chambers, respectively. Then, the sample was mixed with IEF sample buffer (pH 3–7) and loaded on the IEF gel. The IEF Marker 3–10 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a standard for determining the PI of the protein. The running conditions were set at 100 V for 1 h, 200 V for 1 h, and 500 V for 30 min. The gel was fixed in 12% (w/v) trichloroacetic acid (TCA) and stained with Coomassie brilliant blue G-250 (CBB G-250) in accordance with the manufacturer’s manual.
3
Isoelectric Focusing of GFPuv
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Isoelectric focusing of GFPuv was carried out on a PhastSystem using phastgel IEF 3–9 (GE Healthcare Life Sciences) according to the manufacturer’s protocol. Isoelectric points of GFPuv form I and form II were estimated using standard proteins (IEF Marker 3–10, Catalog # 39212-01, ThermoFisher).
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