Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK tagged Sleeping Beauty Transposase (+), using Sleeping Beauty Transposase Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Sleeping Beauty Transposase Antibody recognizes transfected levels of total Sleeping Beauty transposase protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu258 of Sleeping Beauty transposase protein. Antibodies are purified by protein A and peptide affinity chromatography.
Sleeping Beauty Transposase is part of a transposon system designed to allow viral free genetic insertion into vertebrate DNA. The system is composed of two components: a transposable element (transposon) that can carry DNA of interest, and a transposase that cuts and pastes the transposon into the genome. The transposase was identified from a consensus sequence of inactive Tc1/mariner-like transposase DNA sequences from salmonid fish. It was constructed by fusing and modifying two sequences from Atlantic salmon (Salmo salar) and one sequence from rainbow trout (Oncorhynchus mykiss). The transposon,T, was identified from a consensus sequence of extinct Tc-1 like transposons in salmonid fish (Tanichthys albonubes) (1). Further modifications of the system have been made since its initial construction: sequence changes to the transposase to fit better to an improved consensus sequence has increased its efficiency by 100 fold (2).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.