Western Blot with Odyssey Imaging

Proteins were purified, separated and blotted as described with modifications (Frenk et al., 2014 (link)): aged MEP cells were re-suspended in Laemmli buffer containing 100 mM DTT and separated on 15% acrylamide gels, and Odyssey blocking buffer PBS (LI-COR 927 – 40000) was used for antibody hybridisations. Membranes were probed with antibodies listed in Table S7 at given dilutions before imaging on a LI-COR Odyssey 9120 imager. Total protein detection was performed by staining membranes with REVERT Total Protein Stain solution (LiCor 926 – 11010) after antibody detection.

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Cruz C., Della Rosa M., Krueger C., Gao Q., Horkai D., King M., Field L, & Houseley J. (2018). Tri-methylation of histone H3 lysine 4 facilitates gene expression in ageing cells. eLife, 7, e34081.

Publication 2018

Odyssey blocking buffer PBS Odyssey 9120 REVERT Total Protein Stain solution

Acrylamide Antibodies Antibody Buffer Cells Dilutions Gels Hybridisations Laemmli buffer Membranes Odyssey Protein Stain

Corresponding Organization :

Other organizations : Babraham Institute

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Variable analysis

independent variables

Protein purification and separation method (with modifications from Frenk et al., 2014)

dependent variables

Protein expression or abundance (as detected by antibody hybridization and imaging)

control variables

Aged MEP cells
Laemmli buffer containing 100 mM DTT
15% acrylamide gels
Odyssey blocking buffer PBS (LI-COR 927 – 40000) for antibody hybridization
Antibodies listed in Table S7 at given dilutions
LI-COR Odyssey 9120 imager for imaging
REVERT Total Protein Stain solution (LiCor 926 – 11010) for total protein detection

controls

Positive controls: Not specified
Negative controls: Not specified

Annotations

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