|Product Overview :||The kit offers a fast and convenient method to isolate hydrophobic and raft microdomain associated proteins from cells.|
|Applications :||The separated proteins can be used for further experiments such as SDS-PAGE, Western blotting, dot blotting, and immunoprecipitation.|
|Usage :||1 Kit is Sufficient reagents supplied for 80 tests.|
|Storage :||The kit ships on wet ice and it is recommended to store the unopened kit at -20 centigrade. After opening the kit, store the Protease Inhibitor Cocktail for mammalian cell and tissue extracts at -20 centigrade. Store the other components at 2-8 centigrade|
|Kit Components :||• Lysis and Separation Buffer 50 ml
• Wash Buffer for CelLytic Kit 50 ml
• Sodium Chloride, 4 M solution 1.5 ml
• Protease Inhibitor Cocktail for mammalian cell and tissue extracts 1ml
• OptiPrep Density Gradient Medium 100ml
|Materials Required but Not Supplied :||Tissue culture centrifuge tubes,15 ml conical
Dulbecco’s Phosphate Buffered Saline (PBS)
PBS containing 1 mM EDTA
|Preparation :||• Preparation of the Lysis and Separation Working Solution
Always store the Lysis and Separation Buffer refrigerated or on ice. Mix the Lysis and Separation Buffer before use. Prepare the Lysis and Separation Working Solution fresh daily by adding 6 ml of the Protease Inhibitor Cocktail for mammalian cell and tissue extracts to 600 ml of the Lysis and Separation Buffer.
• Modifications of the Lysis and Separation Working Solution and the Wash Buffer ·
The Lysis and Separation Buffer can be diluted with the Wash Buffer (1:1) in order to decrease the volume of the hydrophobic phase, resulting in a more concentrated hydrophobic protein sample.
The Lysis and Separation Working Solution, prepared with Lysis and Separation Buffer diluted with the Wash Buffer, does not significantly affect the protein yield in the hydrophobic phase. · The Lysis and Separation Buffer and the Wash Buffer contain 150 mM NaCl. Some hydrophobic proteins require a higher salt concentration for extraction into the hydrophobic phase. Use the supplied 4 M NaCl solution to adjust the NaCl concentration to the required level for the protein(s) of interest.
|Separation Protocol :||This procedure is suitable for extraction of 10^6 to 10^7 cells. If a larger number of cells are to be used or multiple extractions are run in parallel, adjust the procedure accordingly.
A. Cell collection
For adherent cells steps 1a-3a are required. For cells growing in suspension, begin with step 3a.
1a. Aspirate the growth medium from the tissue culture vessel and wash the cells with Dulbecco’s PBS.
2a. Add 0.1 ml of PBS containing 1 mM EDTA solution per each cm2 of culture area. Incubate until the cells detach. Alternatively, add PBS and scrape the cells using a cell scraper.
3a. Transfer the cell suspension into a 15ml conical tube and collect the cells by centrifugation at 600×g for 5 minutes. Aspirate the supernatant and then store the pellet on ice.
B. Separation of hydrophobic and hydrophilic proteins
1b. Mix the prepared Lysis and Separation Working Solution containing the Protease Inhibitor Cocktail before use. Resuspend 10^6-10^7 cells in 600 ml of ice-cold Lysis and Separation Working Solution. Mix gently by pipetting up and down, and vortex briefly. Transfer the suspension to a microcentrifuge tube.
2b. Incubate the cell suspension on ice for 10 minutes.
3b. Centrifuge the cell lysate in a pre-cooled (4 centigrade) microcentrifuge at 10,000× g for 5 minutes. Transfer the clarified lysate to a new microcentrifuge tube. A 30-50 ml aliquot of the total protein lysate may be saved for further analysis.
4b. Incubate the lysate at 30 centigrade for 3-5 minutes. During the incubation, mix once by inverting the tube. The lysate will turn cloudy during the incubation.
Note: Incubation at 30 centigrade is preferable; however, incubation at temperatures up to 37 centigrade is possible.
5b. Centrifuge the tube in a microcentrifuge at room temperature at 3000×g for 3 minutes. Ensure the centrifuge temperature is higher than 20 centigrade. Do not return the tube to ice after centrifugation. Transfer the upper hydrophilic phase containing hydrophilic proteins to a new microcentrifuge tube. The lower hydrophobic phase is greatly enriched with hydrophobic and raft associated proteins.
6b. In order to remove residual hydrophilic proteins from the hydrophobic phase, the hydrophobic phase may be washed with the Wash Buffer. Add 400 ml of the Wash Buffer to the hydrophobic phase. Mix gently and incubate the tube on ice for 10 minutes. Repeat steps 4b and 5b.
C. Downstream applications
1c. SDS-PAGE electrophoresis Samples from the hydrophilic and hydrophobic phases can be used directly for acrylamide gel electrophoresis. The dye front may appear diffuse but the final protein pattern is not affected. However, some PAGE systems will require the samples to be diluted 5-10 fold to obtain good resolution. Alternatively, the samples can be precipitated with TCA to obtain more concentrated samples. For comparative analysis of protein separation between the phases, it is recommended to normalize the samples loaded on the gel.
2c. Dot blot For fast analysis of an extraction for a specific protein, a dot blot can be performed using 1-2 ml samples.
Before adding the immobilized antibody, dilute the hydrophobic phase 10 to 12-fold with Wash Buffer to make the solution compatible with antibody binding.
4c. Other applications
For applications requiring low salt concentrations, the sample may be dialyzed.