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When extracting proteins from cells the first step is cell culture.
Mouse NIH/3T3 culture cells can be treated with forskolin, using the following method, to be used as a positive control for anti-phospho-CREB, Catalog No. 06-519.
There are numerous methods of cell stimulation and lysis. For a given protein, Millipore's laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Please note that in addition to whole cell lysates there are also membrane and nuclear specific preparations that can be used to isolate and concentrate different cellular components. Please keep in mind that cellular stimulation for extended periods of time must be done using sterile reagents and sterile technique. Always observe the cells microscopically before proceeding with a stimulation or lysis protocol to assure that the cells are viable, not contaminated, and at the desired density is required throughout all protocols.
Cell stimulation and collection will be performed in a tissue culture laboratory/Class II Biological Cabinet.
Since some stimulation conditions can weaken the cell¡¯s attachment to the plate, it is necessary to modify the cell collection technique if this is suspected. This can happen with colcemid treatment and infected/transfected cells.
Cell stimulation and collection will be performed in a tissue culture laboratory/Class II Biological Cabinet.
Cells are generally lysed by treating with detergents, sonication or by repeated freeze and thaw cycles. Extraction conditions release proteases from the cells, thus the need for protease inhibitors in the lysis buffer. Some protease inhibitors, for example, PMSF and benzamidine, have a half-life of only 30 minutes. Add just prior to use.
It is not possible to accurately measure activity of a specific kinase in most cell lysates because of the presence of other kinases that phosphorylate the same substrates, thereby interfering with the assays. Thus, it is critical to immunoprecipitate proteins with kinase activity in order to study signal transduction pathways. After immunoprecipitation, kinase activity is measured in one of two ways. It may be accomplished by subtracting the kinase activity in unstimulated cell lysates from that measured in stimulated cell lysates, or by comparing the kinase activity of the precipitate of the test antibody in stimulated cell lysates with the activity of the precipitate of a corresponding nonspecific antibody (i.e. rabbit or sheep polyclonal IgG/antisera) and/or preimmune sera.
Typically, 500 to 1,000 :g of lysate is used per assay point. An assay performed in triplicate, with controls, uses a large quantity of cell lysate. Different cell lines require the use of growth factors, or stimulants, at different time points to activate the kinase of interest. Kinases may be inactivated by proteases or phosphatases present in cell extracts. Once the conditions for the specific cell line are met, it is very critical that the correct cell lysis buffer be used to prevent this inactivation.
Refer to the Appendix for formulation.
Adherent cells: Add 1 mL of ice cold cell lysis Buffer A/150 mm plate.
The following steps should be carried out in the cold room on ice.
Lysing cells with reducing sample buffer (Lammeli) lacking Bromophenol blue has several advantages. It is quick, simple and reduces concerns that the protein of interest has not been solubilized. The presence of 2% SDS ensures total lysis of the nucleus, and results in the extraction of genomic DNA, which should be sheared by sonication to reduce viscosity. The absence of bromophenol blue enables protein quantitation via the Bradford assay. This lysis procedure is denaturing, and therefore may not be suitable for immunoprecipitation experiments in which the immunoprecipitating antibody recognizes secondary or tertiary protein structure.
For Adherent cells: use 2 to 3 mL of NRSB-MINUS (refer to appendix) per each 150 mm plate depending on cell confluency (i.e., cell density, 2 mL for 80% confluency to 3 mL for a 100% confluent monolayer). Two to 3 mLs of NRSB-MINUS are necessary to sufficiently extract nuclear proteins.
HeLa cells, >109 cells from either spinner culture or 150 mm plates (~40 X 150 mm plates).
Buffer #1 | |
---|---|
HEPES | 10mM |
MgCI2 | 1.5mM |
KCl 1 | 0 mM |
DTT* | 0.5 mM |
Buffer #2 | |
HEPES | 20 mM |
Glycerol | 25 % |
NaCl | 0.42 M |
MgCl2 | 1.5 mM |
EDTA | 0.2 mM |
PMSF* | 0.5 mM |
DTT* | 0.5 mM |
Buffer #3 | |
HEPES | 20 mM |
Glycerol | 20 % |
KCI | 0.1 M |
EDTA | 0.2 mM |
PMSF* | 0.5 mM |
DTT* | 0.5 mM |
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