For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
ORF1p (D3W9O) Rabbit mAb recognizes endogenous levels of total ORF1p protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly162 of human ORF1p protein.
LINE-1, also known as L1, is a non-long terminal repeat (non-LTR) retrotransposon with hundreds of thousands of copies in the human genome (1, 2). Like all non-LTRs, L1 replicates by target-primed reverse transcription (TPRT) (3). The L1 retrotransposon encodes two proteins critical to this process: ORF1p and ORF2p. ORF2p contributes to endonuclease and reverse transcriptase activity, while ORF1p acts as a nucleic acid chaperone that binds RNA (4-8). Many types of cancers have been shown to have L1 insertions within tumor suppressor genes, disrupting their expression and contributing to tumorigenesis (9).
While normally silenced, ORF1p is expressed in many types of cancers (9-12). Recently, it has been found that ORF1p can become phosphorylated and that this event impacts its ability to promote retrotransposition. This creates a novel dynamic to which the host could actively affect L1 replication through various signaling cascades (13).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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