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Tip 1: Working with the extracellular matrix protein rhodamine-labeled fibronectin (cat# FNR01)
When using fluorescently labeled fibronectin as a substrate for matrix metalloproteinases (MPPs), there are reports in the literature that researchers prepare coverslips with a gelatin coating over a poly-L-lysine coating which are then covalently linked by glutaraldehyde fixation. Then fibronectin is applied to the top of that matrix. This precoating with gelatin on top of poly lysine covalently linked by glutaraldehyde creates a favorable orientation for fibronectin since it has a gelatin binding domain. This way of coating coverslips when working with fibronectin could be helpful when studying MPP cleavage of fibronectin. This method of coating is meant to address the concern that MPP-digested fibronectin can remain bound to the slide, making it appear that the MMP hasn’t cleaved fibronectin when in fact it has.
Intermediate filament proteins (IFPs) are sensitive to pH and ionic strength. In fact, it is the manipulation of these parameters that can create polymers or tetramers. Commonly, an IFP is purified into a denaturing buffer containing urea and high concentrations of β-mercaptoethanol. Subsequent sequential steps of dialysis will remove the denaturants and permit renaturation of the protein. Common techniques for working with intermediate filament proteins are listed below:
a) Differential Sedimentation:
Differential sedimentation can be used to remove denatured protein or sediment filaments.
b) Electron microscopy:
Uranyl acetate can be used to visualize IFP filaments by negative stain electron microscopy.
c) Optical absorbance:
Optical density measurements can be used to determine the kinetics of polymerization. A wavelength of 300 to 340 nm is generally applicable.