Western blot analysis of extracts from HT55, SW1463, and HCC827 cells, untreated (-) or treated with PNGase F (+), using CEACAM8 (D2F5U) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock-transfected (lane 1) or transfected with CEACAM8-mycDDK (lane 2), CEACAM6-mycDDK (lane 3) or CEACAM1-mycDDK (lane 4), using CEACAM8 (D2F5U) Rabbit mAb (upper), Myc-tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
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.
CEACAM8 (D2F5U) Rabbit mAb recognizes endogenous levels of total CEACAM8 protein. This antibody does not cross-react with CEACAM1 or CEACAM6 proteins.
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human CEACAM8 protein.
Carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8), also known as CD66b, is a member of the CEA-related cell-adhesion molecule (CEACAM) family (1). CEACAMs bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival (2). CEACAM8 is a single-chain, glycosylphosphatidylinositol (GPI)-anchored, highly glycosylated protein that under normal conditions is expressed almost exclusively on granulocytes (3). Heterophilic interactions of CEACAM8 with other surface molecules, such as CEACAM6, have been shown to be involved in regulating cell adhesion in a calcium-independent manner (4). As such, CEACAM8 has demonstrated utility as a marker of neutrophil and eosinophil activation (5,6) and in pathological conditions is shown to be highly expressed in primary myelofibrosis and acute lymphoblastic leukemias (7,8). Assessment of CEACAM8 positive tumor-infiltrating neutrophils has demonstrated value as a prognostic factor in multiple cancer types (9,10).
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