Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

J Neurosci Methods. 2017 Mar 15;280:64-76. doi: 10.1016/j.jneumeth.2017.02.002. Epub 2017 Feb 13.

Abstract

Background: Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets.

New method: We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer.

Results: Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples.

Comparison with existing method(s): Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation.

Conclusions: This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects.

Keywords: Protein precipitation; Protein solubilization; RIPA buffer; TRIzol; Western blotting.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Blotting, Western / methods*
  • Brain / cytology
  • Brain / metabolism
  • Brain Chemistry*
  • Chemical Precipitation
  • Edetic Acid / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Guanidines / chemistry
  • Male
  • Mice, Inbred C57BL
  • Microglia / metabolism
  • Neurons / cytology
  • Neurons / metabolism
  • Phenols / chemistry
  • Proteins / analysis*
  • Proteins / chemistry
  • Proteins / metabolism
  • Rats, Sprague-Dawley
  • Sodium Chloride / chemistry
  • Sodium Dodecyl Sulfate / chemistry
  • Solubility
  • Temperature
  • Time Factors

Substances

  • Guanidines
  • Phenols
  • Proteins
  • trizol
  • Sodium Dodecyl Sulfate
  • Sodium Chloride
  • Edetic Acid