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.

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.

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.

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).

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.

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.

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.

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 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.

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.

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.