Western blot analysis of extracts from HCC827 and H3255 cells using EGF Receptor (L858R Mutant Specific) (43B2) Rabbit mAb (Biotinylated) and detected with Strepavidin-HRP #3999.
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated EGFR (L858R Mutant Specific) (43B2) Rabbit mAb #3197.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
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.
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* 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: 266
EGFR (L858R Mutant Specific) (43B2) Rabbit mAb (Biotinylated) detects endogenous levels of EGFR mutant L858R protein. The antibody may cross-react with wildtype EGFR and other HER family members when highly over-expressed. Careful titration of this antibody may be required to obtain optimal specificity.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to L858R mutant sequence of human EGF receptor.
The epidermal growth factor (EGF) receptor is a 170 kDa transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Research studies have shown that somatic mutations in the tyrosine kinase domain of EGF receptor (EGFR) are present in a subset of lung adenocarinomas that respond to EGFR inhibitors, such as gefinitib and erlotinib (1-3). Two types of mutations account for approximately 90% of mutated cases: a specific point mutation, L858R, that occurs in exon 21 and short in-frame deletions in exon 19 (4,5). The most frequent exon 19 deletion is E746-A750, accounting for 90% of lesions at this site, although some rare variants occur.
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.