Qproteome Cell Compartment Kit

For fractionation of proteins according to cellular location

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Qproteome Cell Compartment Kit

Cat. No. / ID: 37502

For up to 10 subcellular fractionations: Extraction Buffer, Protease Inhibitor Solution, Benzonase®.

The Qproteome Cell Compartment Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

Eukaryotic cells are complex, well-ordered, and highly structured systems. The Cell Compartment Kit is designed for fast and easy subcellular fractionation of intact eukaryotic cells and tissue. By sequential addition of different extraction buffers to a cell pellet, proteins in the different cellular compartments can be selectively isolated.

Performance

Use of the Qproteome Cell Compartment Kit allows simple and effective cell compartment-specific localization of marker proteins from isolated cells (see figure "" and to "") or from tissue samples (see figure "").

See figures

Specific separation of marker proteins.

Localization of proteins in cells under different growth conditions.

Localization of proteins in tissue samples.

Principle

The kit contains 4 Extraction Buffers which enable the sequential isolation of proteins associated with the cytosol, membranes, nucleus, and cytoskeleton from cell lysates or tissues.

Procedure

The 4 Extraction Buffers are added sequentially to a cell pellet and the respective fractions are isolated by centrifugation (see flowchart).

In an alternative protocol for tissues, samples are simultaneously homogenized and disrupted using the TissueRuptor before being processed as for the cell pellet.

See figures

Flowchart

Applications

Subcellular fractionation of proteins can be used for the enrichment of low-abundance species; to define the subcellular localization of enzymes, regulatory, and structural proteins; and for monitoring of compartmental redistribution of biomolecules under basal and stimulated conditions.

Supporting data and figures

Localization of proteins in cells under different growth conditions.

Micrographs (upper panels) of HeLa cells [A] under normal growth conditions and [B] after 4 hour incubation with staurosporine, an inducer of apoptosis. Lower panels show western blots prepared after cell lysates were processed using the Qproteome Cell Compartment Kit. The blots were probed with anti-cytochrome c antibody. It is known that during apoptosis cytochrome c is translocated from the intermembrane space of mitochondria to the cytosol, a process that is reflected in the detection of cytochrome c in the cytosolic fraction of apoptotic cells.

Specifications

Features	Specifications
Applications	SDS-PAGE, mass spectrometry
Fractions isolated	Four fractions
Start material	Cell lysate
Sample size	~5 x 10e6 cells
Binding capacity/yield	Varies
Species	Eukaryotes

Resources

For the subcellular fractionation of proteomic
samples

FAQ

The Qproteome Cell Compartment Kit, the Qproteome Nuclear Protein Kit, the Qproteome Mitochondria Isolation Kit, and the PhosphoProtein Purification Kit are compatible with tissue samples. The respective protocol can be found in the respective handbook.

The Qproteome Mammalian Protein Prep Kit is also compatible with tissue, there is the QIAGEN Supplementary Protocol: Purification of protein from animal tissues using the Qproteome Mammalian Protein Prep Kit and the TissueRuptor™ available. For all these protocols the TissueRuptor is used.

FAQ ID -755

No, none of the buffers of the Qproteome Cell Compartment Kit contain reducing agents.

FAQ ID -824

Acetone precipitation recommended in the protocols for the Qproteome Protein Fractionation Kits leaves most proteins in a native state. Some proteins may partially denature when precipitating with acetone.

FAQ ID -807

We have not tested immunoprecipitation on protein fractions from the Qproteome Cell Compartment Kit. However, since Buffers CE1, CE2 and CE3 are native buffers, we would expect that they would work for this application. It might be a good idea to dilute the fractions 1:1 with a buffer already tested for immunoprecipitation to adjust to IP conditions. Buffer CE4 is strongly denaturing, so the final protein fraction 4 may not be used for immunoprecipitation.

FAQ ID -855

The following protocol is suitable for concentrating and desalting protein samples for downstream applications such as 2D-PAGE:

Add four volumes of ice-cold 100% acetone to the protein fraction and incubate for 15 minutes on ice
Centrifuge for 10 minutes at 12,000 x g in a pre-cooled microcentrifuge at 4°C
Discard the supernatant and air dry the pellet
Resuspend the pellet in an appropriate sample buffer required for the downstream application

You will find this protocol also in the handbooks for QIAGEN's QProteome Protein Fractionation Kits.

FAQ ID -1035

Endosomal, microsomal and lysosomal proteins will be present in membrane protein fraction 2, as organelles remain intact after addition of Buffer CE1 during the first fractionation step with the Qproteome Cell Compartment Kit.

FAQ ID -808

Soluble mitochondrial proteins will be present in membrane protein fraction 2 when using the Qproteome Cell Compartment Kit.

FAQ ID -839