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Absolute quantification of transcription factors in human erythropoiesis using selected reaction monitoring mass spectrometry.

STAR protocols (2020-12-31)
Mark A Gillespie, Carmen G Palii, Daniel Sanchez-Taltavull, Theodore J Perkins, Marjorie Brand, Jeffrey A Ranish
ABSTRACT

Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass-spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage. For complete details on the use and execution of this protocol, please refer to Gillespie et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Millipore
Benzonase® Nuclease, Purity > 90%
Sigma-Aldrich
myo-Inositol, ≥99%
Sigma-Aldrich
Iscove′s Modified Dulbecco′s Medium, liquid, sterile-filtered, With sodium bicarbonate, without L-glutamine, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis
Sigma-Aldrich
Folic acid, ≥97%
Sigma-Aldrich
1-Thioglycerol, liquid, BioReagent, suitable for cell culture, ≥97% (titration)