Geoderma Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Andrew J. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions. Li, C. Modified universal buffer does not necessarily maintain soil enzyme assay pH. Biol Fertil Soils 57, — Download citation. Received : 13 January Revised : 13 May Accepted : 18 May Published : 08 June Issue Date : August 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. Skip to main content. Search SpringerLink Search. Abstract Buffers are widely used when performing soil enzyme assays, either to measure specific soil enzyme activities based on a standard protocol or to determine the pH optima of a soil enzyme. References Acosta-Martinez V, Cano A, Johnson J Simultaneous determination of multiple soil enzyme activities for soil health-biogeochemical indices.
Margenot Authors Chongyang Li View author publications. View author publications. Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information. Rights and permissions Reprints and Permissions. About this article. Cite this article Li, C. However, there is a lack of systemic evaluation of the efficacy of RIPA buffer in terms of extraction efficiency and completeness of extracted protein profiles.
This mini review focuses on potential problems of using RIPA buffer for total protein extraction and its general use in downstream experiments. Classical RIPA buffer is comprised of low concentration of sodium dodecyl sulfate SDS, a denaturing detergent , deoxycholate for disruption of protein-protein interactions and other components. Generally, only RIPA-soluble fraction is used for downstream experiments. RIPA-insoluble fraction is discarded. It is found that certain proteins are more soluble in RIPA buffer than others.
In recent years, more and more researchers have paid a closer attention to the protein components found in RIPA-insoluble fraction and their effects on experimental results and overall data interpretation.
Bai and Laiho extracted proteins from Hela cell nucleoli by RIPA buffer and found that the protein profiles of soluble and insoluble fraction are quite different indicating the protein loss is not proportional [4]. Mukhopadhyay et al. Wang et al. Li [7] compared protein profiles of RIPA-soluble and RIPA-insoluble fraction of mouse splenocytes and liver tissues and a spin-column based commercial kit and found that protein profiles of RIPA-insoluble fraction are similar but not identical to those found in RIPA-soluble fractions.
The proteins lost to the insoluble fraction cover the whole spectrum of protein profiles and the details of protein species found in different RIPA-insoluble fractions vary from sample to sample.
The protein loss appears to be unpredictable in different samples. However, the commercial kit is much faster and yields more complete protein profile because there is no insoluble fraction involved. Ngoka [8] reported a side by side comparison of protein profiles of RIPA-soluble and RIPAinsoluble fractions from several groups of breast cancer samples by mass spectrometry.
In other word, many high molecular weight proteins are lost to the RIPA-insoluble fractions. It was also shown that nearly all extracellular matrix proteins ECM and many cytoskeleton proteins were found in RIPA-insoluble fractions.
These results indicate that the protein profile extracted by RIPA buffer is incomplete and somewhat biased due to protein loss to the insoluble fraction. A comprehensive analysis of critical factors affecting quantitative immunoblotting was reported recently [12]. The transcription factor GATA-2 and adhesion molecule B-catenin were also present in the insoluble fraction. Another important founding from this study is that sample preparation methods significantly impact the experimental results.
Janes [12] compared the effects of cell lysates derived from NP, RIPA buffer and Laemmli buffer on detection of cleaved form of caspase-8 by Western blotting and found that it was only detected in the cell lysate prepared with Laemmli buffer and it was not detectable in RIPA buffer-derived cell lysate indicating that RIPA buffer is not suitable for this type of analysis.
Cell lysate from RIPA buffer extraction was found to artificially increase the activity of certain protein kinases. The in vitro protein kinase activity derived from RIPA buffer lysate of colon cancer cells was found to be elevated five-seven folds as compared to the activity from the same cells using NP as cell lysis reagent [13].
Zapata et al. These results strongly suggest that great precaution must be taken for data interpretation when RIPA buffer is used. As discussed above, total protein extraction using RIPA buffer suffers many disadvantages because of incomplete protein extraction.
The loss of protein in the extraction process would result in altered protein profiles in terms of total protein species, ratio between different protein species and activity of certain proteins. If the protein lose is consistent and predictable then the choice of sample preparation methods would not be critical.
Due to the loss of proteins into the RIPA insoluble fraction, minimize the adverse effects is a challenging task that generally involves careful evaluation and selection of different protein extraction methods.
An ideal total protein extraction is to obtain a protein profile that faithfully reflects all protein species present in a given sample with a ratio that is a reflection of true protein ratio present in the sample.
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