Question 1: Can HiLyte Fluor&trade, 647-labeled tubulin (Cat. # TL670M) be used to monitor tubulin dynamics in living cells?
Answer 1: Yes, all of Cytoskeleton&rsquo, s fluorescently-labeled tubulins, including TRITC rhodamine-tubulin can be micro-injected into cells to study tubulin localization and dynamics in living cells. Please see the brief protocol on the product datasheet (Cat. # TL670M and these papers for guidance on micro-injecting cells with fluorescently-labeled proteins (Smilenov et al., 1999. Focal adhesion motility revealed in stationary fibroblasts. Science. 286, 1172-1174, Lopez-Lluch et al., 2001. Protein kinase C-d C2-like domain is a binding site for actin and enables actin redistribution in neutrophils. Biochem. J. 357, 39-47, Lim and Danuser, 2009. Live cell imaging of F-actin dynamics via fluorescent speckle microscopy (FSM). J. Vis. Exp. 30, e1325, DOI: 10.3791/1325,
Question 2: What is the best way to store HiLyte Fluor&trade, 647-labeled tubulin to maintain high activity?
Answer 2: The recommended storage condition for the lyophilized tubulin product is 4°, C in the dark with desiccant to maintain humidity at <, 10%. Under these conditions the protein is stable for 6 months. Lyophilized protein can also be stored desiccated at -70°, C where it will be stable for 6 months. However, at -70°, C the rubber seal in the lid of the tube could crack and allow in moisture. Therefore we recommend storing at 4°, C. If stored at -70°, C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. After reconstituting the protein as directed, the concentrated protein in G-PEM buffer should be aliquoted, snap frozen in liquid nitrogen and stored at -70°, C (stable for 6 months). NOTE: It is very important to snap freeze the tubulin in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
If you have any questions concerning this product, please contact our Technical Service department at infohoelzel.de