10μg of C-terminal biotinylated histone H3 1–21aa unmodified, H3R2me1, H3R2me2s, or H3R2me2a peptide (Anaspec) were immobilized on 20μL Streptavidin sepharose resin (Thermo) in 200μL pulldown buffer (50mM Tris-Cl pH 8.0, 150mM NaCl, 5mM β-mercaptoethanol). The peptide-bound resin was washed three times with 500μL pulldown buffer then incubated with 10μM WDR5 full-length protein in 200μL buffer for 1hr with end-over-end rotation at 4°C. The resin was washed 8 times with 500μL buffer and eluted with 20μL 2X Laemelli Buffer and heat denatured at 90°C for 5 minutes. 15μL of eluate was loaded on 15% SDS-PAGE and visualized by Coomassie stain.
Streptavidin sepharose resin
Manufactured by Thermo Fisher Scientific
Streptavidin sepharose resin is a solid support material used for protein purification and affinity-based separation techniques. It consists of streptavidin, a tetrameric protein with a high affinity for biotin, immobilized on a sepharose matrix. This resin can be used to capture and isolate biotinylated proteins, nucleic acids, or other biomolecules from complex samples.
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Lab products found in correlation
2 protocols using streptavidin sepharose resin
1
Histone H3 Peptide Pulldown Assay
2
Histone H3 Peptide Pulldown Assay
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10μg of C-terminal biotinylated histone H3 1-21aa unmodified, H3R2me1, H3R2me2s, or H3R2me2a peptide (Anaspec) were immobilized on 20μL Streptavidin sepharose resin (Thermo) in 200μL pulldown buffer (50mM Tris-Cl pH 8.0, 150mM NaCl, 5mM β-mercaptoethanol). The peptide-bound resin was washed three times with 500μL pulldown buffer then incubated with 10μM WDR5 full-length protein in 200μL buffer for 1hr with end-over-end rotation at 4°C. The resin was washed 8 times with 500μL buffer and eluted with 20μL 2X Laemelli Buffer and heat denatured at 90°C for 5 minutes. 15μL of eluate was loaded on 15% SDS-PAGE and visualized by Coomassie stain.
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