Cell Adherence Inhibition Assay (LTI)
General protocol--Either monoclonal antibody or RGD peptide is added along with the cells during the standard adhesion assay, and the inhibitory effect can thus be semi-quantitated.
ECM Cell ELISA (LTI)
This is general protocol. Specific antibodies to integrins are added to pelleted cells and allowed to bind to the cell surface. The nonbound antibody is washed away and an enzyme conjugated second antibody is added to the re- pelleted cells.The bound second antibody is then detected by the addition of an enzyme-specific substrate and the plate or tubes are read spectrophotometrically. This assay can be used as a screening assay for all integrins for which antibodies are available.
ECM Direct ELISA (LTI)
This procedure can be used for detection of specific proteins or for the titration of an antibody. A protein or mixture of proteins is coated on a polystyrene ELISA plate, and antibody is allowed to bind to the protein. The bound antibody is detected by the addition of a second antibody conjugated to an enzyme and an enzyme substrate with color detector is added. The relative intensity of the color developed is proportional to the original concentration of protein coated on the plate and to the concentration of antibody bound to coated antigen.
ECM Immunoprecipitation (LTI)
Integrin receptors on cell surfaces are labeled with biotin; the receptors are then extracted from the membranes by detergent treatment and saved for immunoprecipitation. Integrin-specific antibodies are bound to Sepharose beads coated with anti-IgG and added to the biotinylated cell extract. Bound receptors are run on an SDS-PAGE, transferred to nitrocellulose, and detected by the addition of streptavidin-enzyme conjugate followed by substrate.
ECM Protocols Western Blot (LTI)
A mixture of protein is separated electrophoretically by SDS-PAGE, and the individual protein bands are transferred to nitrocellulose paper. Specific antibody is then used to probe for any of the bands it might bind to. The bound antibody is then detected by the addition of a second antibody against the immunoglobulin species contained in the first antibody. This second antibody is labeled with an enzyme, and the specific protein band can then be visualized by the addition of an enzyme substrate containing a color developer.
Fibrinogen - Purification (The Cell Biology and Cytoskeleton Group, HMS)
Isolation of Fish Fibronectin from Fish Fibrinogen (The Cell Biology and Cytoskeleton Group, HMS)
Plasminogen Isolation (The Cell Biology and Cytoskeleton Group, HMS)
Prothrombin - Activation (The Cell Biology and Cytoskeleton Group, HMS)
Prothrombin - Purification (The Cell Biology and Cytoskeleton Group, HMS)
Thrombin - Preparation from Fish (The Cell Biology and Cytoskeleton Group, HMS)
"Cells on Gels" (The Cell Biology and Cytoskeleton Group, HMS)
This protocol describes method for culture cell on a thin polyacrylamide-based,collagen-coated flexible substrate. By maintaining a constant total concentration of acrylamide while usr/localying the concentration of bis-acrylamide, it's able to obtain a series of chemically identical substrates with a wide range of flexibility. By usingimaging techniques, cells' response to differences in substrate flexibility can be detected.