Western blot analysis of extracts from various cell lines using BIRC6 (D8B5) Rabbit mAb.
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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.
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
BIRC6 (D8B5) Rabbit mAb recognizes endogenous levels of total BIRC6 protein. Several bands of unknown origin between 120 and 500 kDa are detected in some cell lines. It is possible that these bands are degradation products of BIRC6.Species Reactivity:
Human, Mouse, Rat, Hamster, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp147 in human BIRC6 protein.
BIRC6/BRUCE/APOLLON is a member of the inhibitor of apoptosis protein (IAP) family. BIRC6 is a huge 530 kDa membrane-associated protein with a single survivin-like baculoviral IAP repeat (BIR) domain at the amino terminus, and a ubiquitin-conjugating enzyme domain at the carboxy terminus (1-3). Several research studies support the notion that BIRC6 functions as a dual regulator of cell death and cell division (4-6), and BIRC6 is the only essential BIR-containing protein in mammalian cell growth and development (4,7). Research studies have documented the overexpression of BIRC6 in colon cancer stem cells and in other cancer cell lines (8,9). BIRC6 inhibits apoptosis by either 1) binding to and inhibiting caspases (10) or 2) ubiquitinating the IAP antagonist SMAC and the apoptosis initiator caspase 9, thereby targeting these proteins for proteasomal degradation (4,5). BIRC6 itself is regulated by ubiquitination and proteasomal degradation upon stimulation of apoptosis (7,11).
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