Prior to SEC-MALLS runs, p300 variants were acetylated and deacetylated using p300 HAT or SIRT2 as described previously26 (link). The reactions were analyzed by liquid chromatography-mass spectrometry (LC-MS) as done previously52 (link). Size-exclusion chromatography was performed at a flow rate of 0.5 ml min-1 on a Superdex 200 Increase 10/300 GL column equilibrated in SEC-MALLS buffer (20 mM HEPES, 300mM NaCl, 5μM ZnCl2, 0.5mM TCEP) at 21 °C. A 50 μl sample of p300 at 2 mg ml-1 was injected onto the column and multi angle laser light scattering was recorded with a laser emitting at 690 nm using a DAWN-EOS detector (Wyatt TechnologyCorp. Santa Barbara, CA). The refractive index was measured using a RI2000 detector (Schambeck SFD). The molecular weight was calculated form differential refractive index measurements across the center of the elution peaks using the Debye model for protein using ASTRA software version 6.0.5.3.
Protocol detail
SEC-MALLS Analysis of Acetylated p300
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Buffer
Hepes
Light
Liquid chromatography
Mass spectrometry
Nacl
P300
Protein
Sirt2
Size exclusion chromatography
Tcep
Corresponding Organization :
Other organizations : European Molecular Biology Laboratory, Max Planck Institute for Biophysical Chemistry, University of Leicester, Institut pour l'avancée des biosciences, Inserm, Université Grenoble Alpes, Centre National de la Recherche Scientifique, Institut de Biosciences et Biotechnologies, Physiopathologie et Epidémiologie des Maladies Respiratoires, Washington University in St. Louis
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Variable analysis
independent variables
- Acetylation and deacetylation of p300 variants using p300 HAT or SIRT2
- Molecular weight of p300 variants measured by SEC-MALLS
- Refractive index of p300 variants measured by RI2000 detector
- Flow rate of 0.5 ml min^-1 on a Superdex 200 Increase 10/300 GL column
- SEC-MALLS buffer composition (20 mM HEPES, 300 mM NaCl, 5 μM ZnCl2, 0.5 mM TCEP)
- Temperature of 21 °C
- Sample volume of 50 μl with p300 concentration of 2 mg ml^-1
- Not explicitly mentioned
- Not explicitly mentioned
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