This experiment is designed to identify lipid binding proteins and to analyze lipid protein interaction in a defined membrane environment of lipid sles proteins of cell homogenate are incubated with the freshly prepared lipid vesicles from phosphate seine and ceramide. The reaction mixture is then supplemented with an Xin V conjugated magnetic beads to anchor the protein associated with the lipid vesicles to the magnetic beads. Next protein lipid vesicles are purified by magnetic activated cell sorting.
To identify the lipid bound protein demonstrated results, use SDS page Western blotting to show lyx purification of endogenous and recombinant atypical PKC and a specific ceramide binding domain expressed in MDCK cells. The main advantage of this novel technique over existing methods like Eliza or plasma resonance spectroscopy like surface binding, is that the lipid protein interaction is achieved in a membrane like environment and that the binding proteins can be purified and analyzed. The implications of this technique extend toward therapy and diagnosis of diseases involving lipids because in these diseases, the physiological lipid protein interaction is impaired.
For some of these diseases, the protein targets are not even known and need to be identified To design a lipid-based therapy, Begin with lipid sles prepared from a dried mixture of equimolar, amounts of phosphatidyl seine and C 16 ceramide in 100 microliters of sical buffer. Sonicate for one hour after adding 300 microliters of iCal buffer, supplemented with 0.1 millimolar manganese chloride centrifuge the samples at 12, 000 G for 20 minutes at four degrees Celsius, then resuspend the pellet of large lipid vesicles in 100 microliters of sical buffer. Add one nanomolar of vibrant cm DII fluorescent dye and incubate at 37 degrees Celsius for one hour to facilitate visualization of the SLE fraction after max separation, recover a pink pellet of vibrant cm dai stained phosphate, a Syrian ceramide sles by centrifugation and Resus spend in 100 microliters of tris buffer.
Next, to prepare a detergent free cell lysate sonicate cells in 300 microliters of hypertonic buffer of 10 millimolar tris, hydrogen chloride containing protease and phosphatase inhibitors and centrifuge. To remove membranous debris, now add the clear cell lysate to the lipid physical suspension and incubate the mixture for two hours at four degrees Celsius. Since cell lysates may contain endogenous membranes with phosphate aerin, it is recommended to clear the lysate from these membranes by additional centrifugation steps.
Supplement the reaction mixture with 20 microliters of an XNV binding buffer and 50 microliters of a solution containing magnetic beads conjugated to an XNV incubate for one hour at four degrees Celsius, perform the magnetic activated cell sorting as recommended by the manufacturer's protocol. Next, monitor the vibrant CM DII fluorescence in the flow through and elution fractions using a microplate fluorescence reader to determine the presence and quantity of lipid les in an antibody competition assay. To verify the specificity of the binding reaction of A PKC for the ceramide phospho Seine vesicles incubate one microgram of anti P KC Zeta rabbit polyclonal antibody with a cell lysate for one hour at four degrees Celsius, followed by incubation with the lipid vesicles.
Continue with SDS page and immuno blotting analysis of the protein binding to the ceramide phosphate serin sles in the max eluate for the in vitro reconstitution of a lipid protein polarity complex begin with a pink pellet preparation of vibrant cm DII stained phosphate serian ceramide vesicles as shown earlier, reus suspend the vesicles in 100 microliters of tri buffer, supplemented with 100 micromolar gamma S GT P one millimolar, GDP, and a combination of recombinant proteins of interest. Then incubate the reaction at four degrees Celsius for three hours. Next, add five microliters of a 20 x stock solution of an X in V buffer and 50 microliters of an X in V conjugated magnetic beads.
Incubate the mixture under gentle agitation for another 30 minutes at four degrees Celsius. Continue to perform the NX NV magnetic activated cell sorting as recommended by the manufacturer's protocol to facilitate precipitation. Supplement of the elution fraction with 10 micrograms of pure e albumin.
Concentrate the proteins by weyl flue precipitation. The bottom phase consists of chloroform methanol with enriched vibrant cm dii. The brownish interphase contains the magnetic beads and precipitated protein.
Quantify the ellu lipid vesicles by detection of pink, vibrant cm dii in the organic chloroform methanol phase of the weyl flu reaction. Use equal amounts of lipid vesicles to normalize the amounts of protein between samples and analyze by SDS page. Immuno blotting.
A detergent free lysate of MDCK cells expressing full length PKC Zetta C terminally linked to a green fluorescent protein or a ceramide binding domain. In the C terminus of pkc. Zetta was incubated with phospho seine.
Ceramide vesicles after elucian of the max column, proteins were analyzed by immuno blotting. The ceramide binding fragment C 20 Zeta, EGFP and full length P KC Zeta were recovered from the EIT of the max column, consistent with the specificity and efficacy of the lyx procedure. The negative control EGFP, which did not bind to phosphate, our Syrian ceramide vesicles was not detectable in the eit.
When Linx is complimented with other methods like SDS page and western blotting or proteomics analysis, additional questions can be answered like which proteins are in the lipid binding protein complex. After its development, LabX has paved the way for researchers in the S single lipid research field to explore ceramide induced apoptosis and cell polarity in neuro stem and epithelial cells.
