A systematic approach to quantitative Western blot analysis.

Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process.

Antibody validation for Western blot: By the user, for the user.

Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.

Quantitative Analysis of Signal Transduction with In-Cell Western Immunofluorescence Assays.

Protein levels and signaling events can be efficiently quantified in many samples with the In-Cell Western (ICW) cell-based assay. This quantitative immunofluorescence method streamlines experimental procedures and data analysis, so hundreds of samples can be processed in parallel with quantitative data output. Cells are cultured in microplates and treated with various drugs or conditions. After fixation and permeabilization of cells in the microplate wells, immunostaining is used to detect target proteins. Secondary antibodies conjugated with IRDye near-infrared (NIR) fluorescent dyes are used for multiplex detection and normalization; cell stains and DNA stains can also be used for cell number normalization. Fluorescent signals reflect the protein expression levels or signaling status of the cell population in each well. ICW assays are a powerful alternative to western blotting. Time-consuming, error-prone steps such as cell lysis, gel electrophoresis, and membrane transfer are eliminated. In situ detection of protein targets in fixed cells provides a relevant cellular context, and enables very rapid and precise quenching of cellular treatments. Results are consistent with western blotting, but precision and reproducibility are enhanced. ICW functional assays are used to analyze protein phosphorylation, monitor the timing and kinetics of signal transduction events, monitor cellular response to agonists and antagonists, and determine IC50 and EC50 values. Modified On-Cell Western (OCW) assays are used for analysis of cell surface proteins, receptor internalization and recycling, and fluorescent ligand binding studies. Here, we describe the basic methodology for In-Cell Western quantitative immunofluorescence assays.

A nonfluorescent, broad-range quencher dye for Förster resonance energy transfer assays.

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum approximately 540 nm), Cy3 (approximately 570 nm), Cy5 (approximately 670 nm), IRDye 680 (approximately 700 nm), IRDye 700DX (approximately 690 nm), or IRDye 800CW (approximately 790 nm). The Förster distance R(0) values are calculated as 41 to 65A for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.

Near-infrared fluorescence detection of ATP-biotin-mediated phosphoprotein labeling.

Detection of phosphoproteins plays an important role in understanding protein function in cellular signalling pathways. Improved methods for identification and quantification of phosphoproteins are research priorities. Near-infrared (NIR) fluorescence detection of a gamma-modified ATP-biotin analog was used to detect protein phosphorylation, using both model kinase substrates and mammalian cell lysates. NIR signal intensity was dependent on substrate and ATP-biotin concentrations.

beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.

beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.

Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.

Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.

Near-infrared fluorescence detection permits accurate imaging of loading controls for Western blot analysis.

Housekeeping proteins are typically chosen as internal loading controls for Western blot analysis because of their high, relatively constant expression. It was previously reported that antibodies against beta-actin did not reliably identify differences in sample loading, and extended antibody incubations caused a failure to discriminate differences in target protein levels. Here, beta-actin and GAPDH were evaluated as loading controls using near-infrared fluorescence. A load-dependent response in signal intensity was observed over a 250-fold range of sample concentrations, with R(2) values as high as 0.9939. Longer antibody incubations continued to detect differences in protein level and load-dependent responses became more linear.

A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.

Protein kinases play important roles in many disease processes and are primary targets for drug development. Because cellular phosphorylation cascades are complex multidirectional pathways, the behavior of a drug in a biochemical enzyme assay may not accurately reflect its performance in the context of a whole cell. We have developed a near-infrared cytoblot assay that can be used to investigate both kinase signaling and effects of kinase inhibitors. Adherent cells were grown in either 96- or 384-well plates. Following stimulation, protein phosphorylation was detected immunohistochemically by simultaneous staining with two primary antibodies: a phospho-specific primary and normalization antibody that recognized either the target protein regardless of phosphorylation status (pan protein) or a housekeeping protein. Secondary antibodies labeled with two spectrally distinct near-infrared dyes were used for visualization. Nuclear staining with TO-PRO-3 was also used in place of the normalization antibody. Normalization for well-to-well variability was accomplished by ratiometric analysis of the two wavelengths. The near-infrared cytoblot was used to analyze phosphorylation of EGFR, Akt, Stat3, MEK 1, and ERK1/2. This assay format was also able to simultaneously assess the phosphorylation of multiple signaling proteins in response to known kinase inhibitors. We observed that the IC50 for the EGFR inhibitor PD168393 was similar for EGFR and Stat3 but was significantly higher for ERK1/2, a downstream modulator of EGFR function. The observation that the receptor and its effectors show different IC50 values for the same inhibitory drug could be important for target selection in drug development.

ERK and MMPs sequentially regulate distinct stages of epithelial tubule development.

Epithelial cells undergo tubulogenesis in response to morphogens such as hepatocyte growth factor (HGF). To organize into tubules, cells must execute a complex series of morphogenetic events; however, the mechanisms that underlie the timing and sequence of these events are poorly understood. Here, we show that downstream effectors of HGF coordinately regulate successive stages of tubulogenesis. Activation of extracellular-regulated kinase (ERK) is necessary and sufficient for the initial stage, during which cells depolarize and migrate. ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. Conversely, the activity of matrix metalloproteases (MMPs) is essential for the late stage but not the initial stage. Thus, ERK and MMPs define two regulatory subprograms that act in sequence. By inducing these reciprocal signals, HGF directs the morphogenetic progression of tubule development.

The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p.

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1-8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.