Htrf® Phospho-total Lysis Buffer: a Universal Alternative to Ripa Lysis Buffers

Cisbio offers a comprehensive line of HTRF phosphoand total protein assays to investigate cell signaling pathways. These versatile, homogeneous assays are suitable for a variety of sample types, including primary cell lines and tumor xenografts. Each HTRF assay includes a common and essential step that relies on the ability of the lysis buffer to release the protein of interest. This protein, either phosphorylated or not, is then detected by an HTRF sandwich immunoassay. This application note compares the use of Cisbio’s HTRF phospho-total lysis buffer with commonlyused RIPA lysis buffers for the analysis of cell signaling pathways in different types of samples. The data demonstrate that HTRF phospho-total lysis buffer is as efficient as RIPA lysis buffers to perform protein extraction and is compatible with both simple and more complex biological samples. Moreover, the HTRF phospho-total lysis buffer delivers higher quality HTRF results than RIPA lysis buffers. Finally the HTRF phospho-total Cisbio lysis buffer is not only suitable for HTRF, but also for other analytical methods such as Western blot and ELISA. Any biochemical investigation of biological events that control cell fate, in more or less advanced relevant models, requires cell membrane solubilization first, then the release of cellular protein content. Of course the accessibility of proteins from the different cellular compartments, whether cytoplasmic or nuclear proteins, is essential. For some time, many research laboratories have utilized RIPA lysis buffer (radioimmunoprecipitation assay buffer) as a stringent enough lysis buffer, capable of liberating proteins from the cytoplasm and from the nucleus. However, there are many closely related formulations of RIPA buffer in use. This may result in variability in scientific data from lab to lab, leading to confusion or misunderstanding. In the course of the development of HTRF phosphoproteins assays, Cisbio has carefully optimized a phospho-total lysis buffer that recapitulates lysis efficacy and maximizes HTRF assay. The aim of this study was to demonstrate that the Cisbio’s HTRF phospho-total lysis buffer is perfectly suited to release cytoplasmic and nuclear proteins. The protein extraction efficacy of the lysis buffer is comparable to different commercially available RIPA buffers when applied to 2D or 3D cell cultures, tumor xenografts or tissues. As expected, the performance of HTRF assays is better with the optimized HTRF phospho-total lysis buffer than with the RIPA buffers. In addition to its suitability for various biological samples, HTRF phospho-total lysis buffer delivers valuable results in other applications such as Western blot and ELISA. Considered together, HTRF phospho-total lysis buffer is an excellent alternative to RIPA lysis buffer for biochemical investigations. introduction BIOLOGICAL MODELS & REAGENTS Cell lines: Hela, BXPC3, HEK293 cells were purchased from ATCC. Tumor xenografts and tissues from mice were kindly provided by Dr. Thierry Chardès, IRCM Montpellier. Innovative Biomimesys® scaffolds for 3D cell culture were kindly provided by the company CELENYS. For more information, visit www.celenys.com HTRF phospho-assays: Cisbio Bioassays, Phospho-AKT (Ser473) [64AKSPEG], Advanced phospho-ERK (T202/Y204) [64AERPEG]; phospho-STAT3 [62AT3PEG]; phospho-CREB [64CREPEG]; phospho-EGFR [64EG1PEG] HTRF phospho-total Lysis buffer: Cisbio Bioassays, #64KL4FDF supplemented with blocking reagent #64KB1AAC RIPA buffers were purchased from: Cell Signaling Technologies (CST) # 9806 (R1), Millipore # 20-188 (R2), Thermo Fisher Scientific # 89900 (R3) and Sigma Aldrich # R 0278 (R4). See Table 1 for buffer composition.