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Detection of nucleic acids on nylon-based filters
technical tips
TIC, - - May 1986
Detection of nucleic acids Iodination of proteins* on nylon.based filters This paper descrbes two fast and sensitive methods for visualizing nucleic acids on nylonbased membranes, routinely used for transfer (southern or northern) and h,/ori"dmtiox The methods are claimed to aUow evaluation oft he transfer process and direct and easy comparison of the hybzidization pattern with the complete nucleic acid p a l m of the preparation. Radiolabeledn~eic acids are not required as markers when sta'mingis performed prior to Southern or northern hy'oridizatio~ A negative stahdng, using the Auto-dye technique described for proteins, results in a sensirive and fast detection of nucleic acids fixed on nylon membranes. Subsequent hybridization treatment does not influence the staining. A positive stainingwith a cationic iron colloid is also described.
A new iodination product Protag-125TM is a solid-phase matrix that is claimed to eliminate the drawbacks of earlier methods of protein iodination. The insolubleparticles of Protag125 support an hnmobiliml glycoudl agent. The reaction is completed in 10 ndn at 0-4°C.
The reaction mix is then decanted or filtered from the panicles to stop the iodination, eliminating the need for harsh reducing agents. The large surface area of the reagent allows efficient protein iodination in a small volume of buffer without previous equilibrium or preparative coating steps. Iodination and subsequent re-
moval of any free~Sl from ten protein samples can be completed in 25-30 min using Protag-125 and the speTM sample preparation system. *Based on informationprovidedby I. T. BakerChemicMCo., 222 Red SchoolLane, e~ipsburg, NJ0SS65, USA.
Saman, E., (1986) (;me Anal Techn. 3, 1-5
117
TIC, - - May 1986
Detection of nucleic acids Iodination of proteins* on nylon.based filters This paper descrbes two fast and sensitive methods for visualizing nucleic acids on nylonbased membranes, routinely used for transfer (southern or northern) and h,/ori"dmtiox The methods are claimed to aUow evaluation oft he transfer process and direct and easy comparison of the hybzidization pattern with the complete nucleic acid p a l m of the preparation. Radiolabeledn~eic acids are not required as markers when sta'mingis performed prior to Southern or northern hy'oridizatio~ A negative stahdng, using the Auto-dye technique described for proteins, results in a sensirive and fast detection of nucleic acids fixed on nylon membranes. Subsequent hybridization treatment does not influence the staining. A positive stainingwith a cationic iron colloid is also described.
A new iodination product Protag-125TM is a solid-phase matrix that is claimed to eliminate the drawbacks of earlier methods of protein iodination. The insolubleparticles of Protag125 support an hnmobiliml glycoudl agent. The reaction is completed in 10 ndn at 0-4°C.
The reaction mix is then decanted or filtered from the panicles to stop the iodination, eliminating the need for harsh reducing agents. The large surface area of the reagent allows efficient protein iodination in a small volume of buffer without previous equilibrium or preparative coating steps. Iodination and subsequent re-
moval of any free~Sl from ten protein samples can be completed in 25-30 min using Protag-125 and the speTM sample preparation system. *Based on informationprovidedby I. T. BakerChemicMCo., 222 Red SchoolLane, e~ipsburg, NJ0SS65, USA.
Saman, E., (1986) (;me Anal Techn. 3, 1-5
117