Therefore, the applicability of this method to the evaluation of steady-state expression of the whole proteome, particularly for undetectable proteins or to quantitative analysis, needs further testing. Performance of the modified TRIzol protocol evaluated by immunoblotting.
It is known that multiple proteins involved in the regulation of key cellular functions are deregulated in many diseases, including cancer. Worthy of note, PARP is a kDa nuclear protein, cleaved by caspase-3 upon apoptotic stimuli, yielding an 85 kDa fragment.
With the modified TRIzol protocol, it was possible to detect the full length and the cleaved fragment, with increased sensitivity compared to other protocols. We and others have used Ponceau S staining as a loading control for western blot [ 14 — 16 ]. This is particularly relevant when experiments require protein separation in low percentage SDS polyacrylamide gels, in which the typical housekeeping controls migrate out of the gel. In addition, it is also important in conditions in which the typical housekeeping proteins are not adequate controls, due to changes in their steady-state expression levels, including during stem cell differentiation [ 14 , 16 ].
In this study, we validated the performance and used Ponceau staining as a normalization control for immunoblot analysis. The closer the slope is to zero, the higher is the concordance between both variables.
Our results provided a mean slope of 0. Steady-state protein expression levels in human colon cancer samples. These mutational events have been reported to occur at different phases of the multistep process of malignant transformation [ 17 ].
However, the loss of p53 function in CC is usually associated with adenoma to carcinoma transition [ 18 , 19 ]. In most cases, the p53 gene mutation gives rise to a stable mutant protein, which tends to accumulate in the cell [ 17 ].
In addition, the loss of functional p53 in adenomas and dMMR carcinomas represents the minority of cases, in contrast to pMMR carcinomas, where p53 is frequently mutated [ 19 , 21 ]. In addition, p53 is critically involved in drug-induced apoptosis, in which it is up-regulated and translocated to mitochondria following HCT colon cancer cells exposure to 5-fluorouracil [ 23 ]. Importantly, p53 levels in adenomas and dMMR carcinomas were much lower than those found in pMMR carcinomas, which might be associated with an attempt to control cell growth, rather than reflect p53 mutations.
The high steady-state levels of p53 found in pMMR carcinomas were possibly related to the accumulation of stable mutant forms of p The present results are in accordance with the literature, and suggest that the loss of functional p53 is indeed involved in the adenoma to carcinoma transition, and that the status of the mismatch repair system is strongly correlated with the frequency of p53 mutations [ 24 ].
In turn, Akt overactivation is typically more common then its overexpression, due mainly to the overexpression of EGFR in this type of cancer [ 27 ]. In addition, the involvement of Akt in sporadic colon carcinogenesis has already been reported as an important early event [ 30 ]. Steady-state protein expression levels in mouse brain samples.
Ponceau S staining was used as loading control. Therefore, the modified TRIzol protocol for protein extraction provides an adequate tool for protein analysis in brain tissue samples, coupled to RNA analysis.
This modified protocol allows higher protein recovery yield, while producing protein patterns comparable to those obtained by other extraction methods, following SDS-PAGE and Coomassie or Ponceau staining.
In addition, we demonstrated the importance of the sample sonication step for efficient ressuspension of the recovered protein pellet. Our modified TRIzol protocol also allowed the detection of post-translational modifications, namely protein phosphorylation, and cellular protein distribution. Furthermore, our modified method allowed the detection and relative quantification of steady-state expression levels of important proteins deregulated in disease conditions, reproducing the current literature.
However, we cannot conclude if our method would be suitable for quantitative protein analysis, nor for mass spectrometry since high concentrations of urea may lead to protein carbamylation.
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Article PubMed Google Scholar. J Clin Invest. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Pedro M Borralho. All authors read and approved the final manuscript. Reprints and Permissions.
BMC Genomics 14, Download citation. Received : 15 July Accepted : 06 March Published : 15 March Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. An abnormal, absorbance maxima at nm indicates phenol contamination Figure 4. With genomic DNA contamination, indicators can be inflated yields on the spectrophotometer and high molecular weight band s or a smear on an agarose gel using electrophoresis Figure 5.
However, there is a greater need for faster handling time, reliability and consistency for ultra-pure RNA. Furthermore, the Zymo-Spin silica-based binding column is uniquely designed to capture unbiased total RNA down to picogram amounts single-cell Figure 7.
Citations: [1] Beintema JJ, van der Laan JM "Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases". A procedure for the isolation of mammalian messenger ribonucleic acid. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. Email Address Password Forgot Password. First Name. Figure 3. Figure 5.
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