Measurement of protein using bicinchoninic acid. the immunoblot. The premise of immunoblotting is simple, but execution is definitely tricky, and there are numerous variations in the method that can impact the outcome (1). Add quantitation to the end of an immunoblot and the difficulty of implementations raises even further. Surprisingly, you will find few objective studies on quantitative immunoblotting in the primary literature (2, 3). Lacking a systematic assessment of key factors, researchers are prone to repeat or reinforce mistakes that others have made before them. Here, I analyze how numerous methodological choices impact the ability to perform quantitative immunoblotting accurately and exactly. The analysis exposed how seemingly small variations affect immunoblot linearity and reproducibility, yielding pseudoquantitative figures that are not directly proportional to the input material. After background subtraction, quantitative immunoblots should strive for zero-intercept linearity: = is the quantified band intensity, is the GW 542573X large quantity of the protein or changes state in the sample, and is a proportionality coefficient. The value of is flexible, but lines with nonzero intercepts indicate errors in background subtraction, and nonlinear associations suggest problems with detection level of sensitivity or saturation. Either scenario will yield fold-change estimations that are skewed relative to Cd86 the true variations among samples. Throughout this work, I systematically modified several experimental guidelines that are often neglected or overlooked when immunoblotting. Many other guidelines were kept fixed: All gels were run as 15-well, 1.5-mm solid, Tris-glycine minigels within the Bio-Rad Protean III platform; all damp GW 542573X electrophoretic transfers were carried out onto low-autofluorescence, 0.45-m polyvinyldifluoride (PVDF) less than altered Towbin conditions (4) (25 mM Tris, 192 mM glycine, 0.0375% SDS, 10% GW 542573X methanol unless otherwise indicated); detection was performed on either a LI-COR Odyssey instrument (for fluorescence detection) or a Bio-Rad ChemiDoc MP gel imager (for chemiluminescence detection); and quantitation of natural 16-bit digital images was implemented with the ImageJ gel analysis plugin (5). Using film to perform quantitative immunoblotting was avoided entirely, because the dynamic range of film is so small that quantitative analysis is virtually impossible (3). Film can make small differences in abundance appear as large differences in band intensity. When saturated, film exposures can also hide sample-to-sample variations in high-abundance proteins such as loading settings. Therefore, throughout this study, all data were acquired as digital images. The diagnostic experiments demonstrated here can be very easily adapted for additional hardware and reagent configurations. Results Sample preparation is a critical element for quantitative immunoblotting The conditions of cell lysis have a profound impact on the proteins that are extracted and the condition in which they may be preserved. For example, lysis of cells or cells with purely nonionic detergents (Triton X-100 or NP-40) causes some proteins to partition into the soluble and insoluble (pellet) fractions after centrifugation. Radioimmunoprecipitation assay (RIPA) buffercontaining dilute sodium dodecyl sulfate (SDS, a denaturing detergent) and deoxycholate (a disruptor of protein-protein relationships)is widely used like a lysis buffer for whole-cell extraction. Nonetheless, RIPA buffer lysis still generates an insoluble portion with major protein constituents from your cytoskeleton and extracellular matrix (6). To test how GW 542573X RIPA lysis conditions affected immunoblotting results, I lysed HT-29 human being colon adenocarcinoma cells in RIPA buffer, boiled the RIPA-insoluble pellet in an equal volume of dithiothreitol-containing Laemmli sample buffer (7), and then immunoblotted for 20 different protein focuses on. As expected, RIPA lysis buffer efficiently solubilized many cytoplasmic proteins [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), warmth shock protein 90 (Hsp90)] and signaling proteins [inhibitor of nuclear factor-B (I), numerous kinases] (Fig. 1A and B). RIPA buffer also extracted the cytoskeletal and cytoskeleton-associated proteins, actin and focal adhesion kinase (FAK). However, tubulin and intermediate filament proteins (lamin A and KRT5) showed substantial losses into the RIPA-insoluble portion (Fig. 1C). Amazingly, RIPA insolubility was not limited to cytoskeletal proteins: The transcription element GATA2 and the cell-cell adhesion protein -catenin were also present in the insoluble portion. In contrast, lysis with GW 542573X Laemmli sample buffer, followed by shearing of the viscous genomic DNA having a high-gauge needle, solubilized proteins that are tightly associated with DNA, such as histones (Fig. 1D). Despite rules of thumb for protein solubility in various lysis buffers (6), these results show that it is best to confirm appropriate solubilization of proteins of interest before embarking on an immunoblot study. Open in a separate windows Fig. 1 Radioimmunoprecipitation assay (RIPA) buffer solubilizes many, but not all, cellular proteins. (A) Examples of proteins that are entirely solubilized (100% in.