Western Blot

Description

Fast and reproducible extraction of subcellular proteomes from mammalian cells

ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from adherent and suspension-grown mammalian cells. The S-PEK takes advantage of the different solubilities of certain subcellular compartments in the four selected reagents. In the case of adherent cells, the procedure is performed directly in the tissue culture dish without the need for cell removal. Cells or the parts of the cells remain attached to the plate during sequential extraction of subcellular compartments, until the appropriate extraction reagent is used. Thus, the early destruction of the cellular structure by enzymatic or mechanical detachment of cells from the tissue culture plate and any mixing of different subcellular compartments is prevented. For suspension-grown cells, extraction starts with gentle sedimentation and washing of the cells. The stepwise extraction delivers four distinct protein fractions from one sample:

– Cytosolic fraction (F1)
– Membrane/organelle protein fraction (F2)
– Nucleic protein fraction (F3)
– Cytoskeletal fraction (F4)

Proteins are obtained in the native state making the S-PEK suitable for many downstream applications such as 1D and 2D gel electrophoresis, immunoblotting, enzyme activity assays, and protein microarrays.

Sample size: 3-5×106 or 25-50 mg tissue.

Description

The ProteoExtract® Transmembrane Protein Extraction Kit (TM-PEK) provides a detergent-free novel chemistry to enable the mild and efficient extraction of transmembrane proteins, such as GPCRs, from mammalian cells and tissues. The fraction enriched in membrane proteins and large protein complexes is directly compatible with enzyme assays, non-denaturing and SDS-based gel electrophoresis, Western blotting and immunoprecipitation. The enriched fraction is appropriate for mass spec-based analysis after a tryptic in-gel digest. The kit contains two different extraction reagents for protein-dependent optimization, protease inhibitor cocktail, and appropriate buffers for processing twenty samples (2-5 x 106 cells or 25-50 mg tissue per sample).

Description

SignalBoost™ Immunoreaction Enhancer Kit is designed to enhance the desired signal in assays such as immunoblotting, dot blotting, and ELISA. This kit significantly improves sensitivity and discrimination when assessing protein targets of low immunoreactivity or low expression levels. Enhancement of signal is especially marked for low affinity antibodies. It replaces conventional antibody diluents with Solution 1, which is used to dilute primary antibodies and Solution 2, which is used to dilute secondary antibodies. No additional steps, materials, or increase of assay time is required.

• Increases signal: minimizes background resulting in high signal to noise ratios
• Versatile: can be used with nitrocellulose or PVDF membranes and is compatible with chemiluminescent or colorimetric detection systems
• Convenient: ready-to-use format, no dilution necessary

Description

YeastBuster™ Protein Extraction Reagent is formulated for a fast, efficient and gentle extraction of active proteins from yeast cells. The reagent avoids harsh conditions of vigorous mechanical or chemical treatment that often result in heat degradation of target proteins. The proprietary formulation utilizes a mix of mild detergent, protein stabilization buffer, and tris(hydroxypropyl) phosphine (THP) reducing agent (THP concentrate provided separately). This powerful combination eliminates the inconsistencies associated with tedious mechanical disruption of yeast cells with glass bead abrasives, ultrasonication and pressure disruption, or enzymatic digestion with β-1,3-glucanase lytic enzymes. The reagent has been tested with Saccharomyces cerevisiae, Pichia pastoris, P. stipidis, and Schizosaccharomyces pombe strains and with plant cells.

In practice, cells are harvested by centrifugation and suspended in YeastBuster. Following a brief incubation, insoluble cell debris is removed by centrifugation, and the clarified extract is ready to use. In addition to greater total protein yields in crude extracts and recovery of enzymatically active protein, the extracts are fully compatible with GST•Bind™ and Ni-NTA His•Bind® immobilized metal affinity chromatography (IMAC) purification methods. The reagent is available in 100- and 500-ml sizes.