Cat. No. / ID: 37502
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.
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.
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.
|applications||SDS-PAGE, mass spectrometry|
|samplesize||~5 x 10e6 cells|
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.
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.
The following protocol is suitable for concentrating and desalting protein samples for downstream applications such as 2D-PAGE: