Tissue Homogenization and Fractionation

All tissues were further dissected
into small pieces and homogenized at 4C using the Precellys 24 homogenizer
in PBS with protease and phosphatase inhibitors (Roche, Indianapolis,
Indiana). The brain tissue that was fractionated (Figure 1E) was prepared differently.
It was homogenized and fractionated using sucrose gradient fractionation
method following a previously published protocol.^{29 (link)} After homogenization, protein concentration was determined
with a Pierce BCA protein assay (Life Technologies, Grand Island,
NY).

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McClatchy D.B., Ma Y., Liu C., Stein B.D., Martínez-Bartolomé S., Vasquez D., Hellberg K., Shaw R.J, & Yates JR I.I.I. (2015). Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice. Journal of Proteome Research, 14(11), 4815-4822.

Publication 2015

Precellys 24 protease and phosphatase inhibitors Pierce BCA protein assay

Assay Brain Inhibitors Phosphatase Protease 24 Protein Sucrose Tissue

Corresponding Organization :

Other organizations : Scripps Research Institute, Institute of Computing Technology, Chinese Academy of Sciences, Salk Institute for Biological Studies, Howard Hughes Medical Institute

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Tissue homogenization method (Precellys 24 homogenizer vs. sucrose gradient fractionation)

dependent variables

Protein concentration

control variables

Temperature (4°C)
Protease and phosphatase inhibitors (Roche, Indianapolis, Indiana)
PBS buffer

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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