Efficient recovery of proteins from multiple source samples after trizol® or trizol®LS RNA extraction and long-term storage

Abstract

BackgroundSimultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol® and TRIzol®LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.ResultsTRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer’s disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at −80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer’s protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer’s protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at −80°C.ConclusionsWe provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer’s disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

Resveratrol Reduces the Proinflammatory Effects and Lipopolysaccharide- Induced Expression of HMGB1 and TLR4 in RAW264.7 Cells

“…Total RNA was extracted from RAW264.7 cells by Trizol reagent (Invitrogen, Corp., Carlsbad, USA), according to the manufacturer's instructions [23,24]. Quantitative determination of RNA was conducted using the GeneQuant pro RNA/DNA Calculator spectrophotometer (Amersham Biosciences, Freiburg, Germany).…”

Anti-inflammatory effect of resveratrol through the suppression of NF-κB and JAK/STAT signaling pathways

“…Cells were seeded in 6-well culture plates at a density of 1 × 10 6 cells/ well and serum-starved for 18 h. Cells were then pretreated with resveratrol for 1 h and stimulated with 1 μg/ml LPS for 4 h. Total RNA was isolated from cells using TRIzol reagent following the manufacturer's instructions [18], and complementary DNA was synthesized using 3 μg of RNA in a reverse transcription reaction from real-time polymerase chain reaction (RT-PCR) kit (Invitrogen, Carlsbad, USA). Quantitative RT-PCR (qRT-PCR) analyses were performed using C1000 thermal cycler (Bio-Rad) with SYBR-Green (Invitrogen).…”

Western Blot Analysis of Cells Encapsulated in Self-Assembling Peptide Hydrogels

“…Urea, a chaotropic salt, was investigated for its ability to disrupt hydrogen bonding (33) and reduce hydrophobic interactions (34), the two key interactions involved in peptide self-assembly into -sheet rich fibers (35). In fact, urea has been noted to increase protein yield following protein precipitation (36) and together with thiourea was shown to significantly improve protein solubilization (37,38). Moreover, it has been shown that urea is an effective agent for solubilizing poorly soluble (hydrophobic) membrane-associated proteins (39), proving more effective than RIPA buffer when tested with small heat-shock proteins (40).…”