Cell binding assay
View Patent ↗The present invention relates to a fluorescent cell binding assay combining pre-labeling and Western blotting. Intact cells are incubated with pre-labelled binders preferably followed by SDS PAGE (sodium dodecylsulphate polyacrylamide gel) separation and Western blotting. More closely, the invention relates to a cell binding assay in which the degree or amount of binding of one or more cell interacting protein or protein component to the cell surface is measured with the ability to correlate the degree of cell binding to the sample load/total number of cells.
1. A method for determining the amount of binding of a cell interacting component to a cell surface structure present in a cell sample, comprising:
a) pre-labelling said cell interacting component with a first dye;
b) incubating said component with desired cells to allow said component to bind to cell surface structures;
c) washing away labelled cell interacting component not bound to the cells;
d) lysing the cells to form a cell lysate;
e) electrophoretic separation of proteins in said cell lysate in a gel, preferably a SDS PAGE gel;
f) transferring proteins from the gel to a Western blotting membrane;
g) Western blotting detection of housekeeping protein;
h) scanning or imaging of the membrane; and
i) measuring signals from the pre-labelled cell interacting component and housekeeping protein.
2. The method of claim 1 , comprising measuring the signals obtained in step h) in relation to total sample load/total no of cells by probing the membrane with labelled (using a different dye than the said first dye used for the pre-labelling of the cell interacting component) binders/antibodies against one or more house-keeping proteins in said cell sample and by relating the signal from the cell interacting component with the signals from the house-keeping protein(s).
3. The method of claim 1 , comprising measuring the signals obtained in h) in relation to total sample load/total no of cells by pre-labelling all proteins in the cell lysate obtained in step d) with another dye than the one used for labelling of the cell interacting component, and by relating the signal from the cell interacting component with total proteins in the cell lysate.
4. The method of claim 1 , comprising measuring the signals obtained in g) in relation to total sample load/total no of cells by post-labeling all proteins on the Western blotting membrane obtained in step f) with another dye than said first dye used for labelling of the cell interacting component.
5. The method of claim 1 , wherein all labelling is made with a fluorescent dye.
6. The method of claim 5 , wherein the dye is a cyanine dye, or any other dye enabling multiplex detection.
7. The method of claim 1 , wherein the binder/antibody targeting house-keeping proteins is labelled/conjugated with a fluorescent dye.
8. The method of claim 1 , wherein the binder/antibody targeting house-keeping proteins is conjugated with horse radish peroxidase (HRP) or alkaline phosphatase (AP) or similar enzyme for subsequent ECL detection.
9. The method of claim 1 , wherein the cell interacting component is Annexin V or any other protein or protein component.
10. The method of claim 1 , wherein the cell interacting component is binding to cell surface of cells or fractions of cells.