Proteins were resolved using SDS-polyacrylamide gel electrophoresis (PAGE). Samples were heated at 80 °C for 5 min to better resolve proteins. The electrophoresis was set for 30 min with 110 V followed by 125 v until the loading dye reached the bottom of the gel (~ 1.5 h) using vertical Mini-PROTEAN® Tetra cell system (BioRad, Catalog # 1658000, Mississauga, Canada). Gels were washed twice with distilled water and treated with Coomasie solution (Acetone: Methanol: distilled water 10:45:45 with 0.25% Brilliant Blue G-250), de stained with de-staining solution (Acetone: Methanol: distilled water 10:45:45) until protein bands were observed. The protein gels were then visualized using Gel DocTM EZ imager system (BioRad, Catalog # 1708270).
Gel doctm ez imager system
Manufactured by Bio-Rad
Sourced in Germany
The Gel Doc™ EZ imager system is a compact, all-in-one system designed for visualization and documentation of nucleic acid and protein gels. The system provides high-quality image capture and analysis capabilities, enabling users to document and analyze gel-based experiments.
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5 protocols using gel doctm ez imager system
1
SDS-PAGE Protein Separation and Visualization
2
RNA Isolation and RT-PCR Analysis
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Total RNA was isolated from cells (1 × 106) by using the NucleoSpinR® RNA Kit II (Macherey-Nagel GmbH, Düren, Germany) in accordance to the manufacturer’s instructions. cDNA was reverse transcribed from mRNA using the RevertAid™ First Strand cDNA Synthesis Kit (VWR International, Darmstadt, Germany) are referred to instruction manual. For PCR (total volume 25 µL per reaction) 1.25 U Taq Polymerase, 1× reaction buffer, 2 mM MgCl2, 2 µM dNTPs (all reagents from VWR International, Darmstadt, Germany) and 100 µM primers (Life Technologies, Darmstadt, Germany) were used. The cycling conditions comprised of an initial denaturation step (5 min at 95 °C), 35 cycles of amplification (30 s 94 °C; 30 s appropriate annealing temperature; 30 s 72 °C) and final elongation (10 min 72 °C). PCR products were separated on a 1% TAE agarose gel. PCR bands were visualized by GelRed™ staining (VWR International GmbH, Darmstadt, Germany) and the GelDocTM EZ Imager System (Bio-Rad, Munich, Germany). Primer pairs used in this study are summarized in Table 1 .
3
Histone Deposition Assay with FACT
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Tetrasomes for histone deposition assays were reconstituted on 87 bp DNA fragments using the salt-dialysis method. Different concentrations (25 nM and 50 nM) of FACT complexes were first incubated with H2A/H2B dimer (100 nM) at 4 °C for 30 min, and then mixed with the constant reconstituted tetrasomes (100 nM) in reaction buffer (10 mM HEPES, pH 8.0, 1 mM EDTA, 60 mM NaCl). The samples were incubated at 30 °C for 1 hr prior to electrophoresis on 5% native PAGE electrophoresis in 0.25 × TBE buffer (22.5 mM Tris, pH 8.0, 22.5 mM boric acid, 0.5 mM EDTA) for 1.5 hr at 80 V. The gels stained with ethidium bromide were scanned by a Gel DocTM EZ Imager system (Bio-Rad), with the uncropped and unprocessed scans of all of the blots shown in the Source Data file.
4
SDS-PAGE Protein Visualization Protocol
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Proteins were resolved using SDS-polyacrylamide gel electrophoresis (PAGE). Samples were heated at 80 0 C for 5 min to better resolve proteins. The electrophoresis was set for 30 min with 110 V followed by 125 v until the loading dye reached the bottom of the gel (~1.5 hour) using vertical Mini-PROTEAN® Tetra cell system (BioRad, Catalog # 1658000, Mississauga, Canada). Gels were washed twice with distilled water and treated with Coomasie solution (Acetone: Methanol: distilled water 10:45:45 with 0.25% Brilliant Blue G-250), de stained with de-staining solution (Acetone: Methanol: distilled water 10:45:45) until protein bands were observed. The protein gels were then visualized using Gel DocTM EZ imager system (BioRad, Catalog # 1708270).
5
FACT Binding Dynamics to Nucleosomes
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Detailed methods are provided in the online version of this paper and include the following: FACT's dual function of attenuating and maintaining the nucleosome structure is obtained through three interactions, interaction between SPT16 and the H2A/H2B dimer, interaction between SSRP1 and the H3/H4 tetramer, and the interaction between HMG and DNA.
for Protein Sciences, Institute of Biophysics, and Chinese Academy of Sciences. We are also indebted to the colleagues whose work could not be cited due to the limitation of space. DNA binding assay 0.1 mg 187 bp 601 DNA fragments were incubated with increasing concentrations of FACT proteins in DNA binding buffer (20 mM Tris, pH 7.5,75 mM NaCl,1 mM EDTA,15% glycerol) at 25 C for 1 hr. The reaction products were separated by 5% native PAGE electrophoresis in 0.25 3 TBE buffer (22.5 mM Tris, pH 8.0, 22.5 mM boric acid, 0.5 mM EDTA) for 1.5 hr at 80V. The gels were stained with ethidium bromide and scanned on a Gel Doc TM EZ Imager system (Bio-Rad). The DNA intensity of each band was quantified with Gel-Pro Analyzer software (Media Cybernetics) and used to calculate the percentage of unbound DNA.
for Protein Sciences, Institute of Biophysics, and Chinese Academy of Sciences. We are also indebted to the colleagues whose work could not be cited due to the limitation of space. DNA binding assay 0.1 mg 187 bp 601 DNA fragments were incubated with increasing concentrations of FACT proteins in DNA binding buffer (20 mM Tris, pH 7.5,75 mM NaCl,1 mM EDTA,15% glycerol) at 25 C for 1 hr. The reaction products were separated by 5% native PAGE electrophoresis in 0.25 3 TBE buffer (22.5 mM Tris, pH 8.0, 22.5 mM boric acid, 0.5 mM EDTA) for 1.5 hr at 80V. The gels were stained with ethidium bromide and scanned on a Gel Doc TM EZ Imager system (Bio-Rad). The DNA intensity of each band was quantified with Gel-Pro Analyzer software (Media Cybernetics) and used to calculate the percentage of unbound DNA.
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