Gel shift

S. E., The Gel Shift Assay for the analysis of DNA-protein interactions. In DNA-Protein Interactions Principles and Protocols. Methods in Molecular Biology Vol. 30, Kneale G.G. (Ed.), Humana Press Inc. Totowa, NJ, 1994. 

To perform gel-shift selection experiments, prepare a native gel, containing 3% acrylamide (stock 38.5% acrylamide and 1.5% bis-acrylamide) and lx TBE, adjusted to pH 7.4. 

Amplify eluted cocaine-displacable RNA + proceed with gel-shift selection... 

The techniques called mobility shift or gel shift assay can be considered a first step in this direction. These are widely used in molecular biology to detect interactions between regulatory proteins for gene expression and specific sequences of polynucleotides (21-23). 

Mixing different cross-linkers (19b and 19c) yielded systems with a strong gel-weak gel transition, rather than a distinct sol-gel shift. When the concentration of each of the cross-linkers was above the critical percolation threshold, the kinetically slower cross-linker (19c) dictated the gel properties. Upon addition of enough of a competitive binding additive, such as DMAP, to drop the concentration of the active cross-linking units below their individual percolation thresholds but still allowing the total amount of both active cross-linkers (19b and 19c) to be above the percolation threshold results in a gel whose properties are now controlled by the kinetically faster cross-linker (19c). 

Kay Was the VRI that you used in the gel shift assays bacteriaUy expressed ... 

Van Gelder Do you have any idea about the kinetics of the gel shift How long do you have to keep that extract in the light before you see the shift What is the relationship of this to the in vivo kinetics of phase shifting ... 

Other applicable selection methods include immunoprecipitation [24] and gel-shift assays [25]. Generally, a method must be found that enables the separation of unbound from bound RNA. 

The problem with gel-shift assays concerns the fact that the interactions under investigation must be very strong (low kQff rates). Weakly interacting binding partners dissociate too quickly during electrophoresis and prevent determination of the Ky> value. 

Fig. 8.6. By utilizing a streptavidin-coupled immobilized matrix or gel-shift PAGE, the affinity tag biotin allows the RNA catalysts to be captured and separated from the noncatalytic RNA.

In addition to Ras, a number of other proteins are known to be substrates for FTase (many of unknown identity or function, determined by 2-D gel shift experiments in the presence and absence of FTI) these may also play a part in determining relative susceptibility to FTase inhibition.46 In particular, RhoB, a farnesylated protein involved with the formation of actin stress fibers and cytoskeletal organization, has been implicated in the growth-inhibitory consequences of FTase inhibition.47,48 Another study using the FTI 19 found that growth inhibition in soft agar of a panel of human tumor cell lines correlated with the mutational status of the H-and N-Ras proteins, but not with K-Ras mutations.49 In this study also, cells with genetic defects in addition to ras were sensitive to FTase inhibition. 

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