Western blot analysis of extracts from various cell lines using Bok (D7V2N) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Bok (hBok-Myc/DDK; +) using Bok (D7V2N) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (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.
Bok (D7V2N) Rabbit mAb recognizes endogenous levels of total Bok protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val88 of human Bok protein.
The Bcl-2 family consists of a number of evolutionarily conserved proteins containing Bcl-2 homology domains (BH) that regulate apoptosis through control of mitochondrial membrane permeability and release of cytochrome c (1-3). Four BH domains have been identified (BH1-4) that mediate protein interactions. The family can be separated into three groups based upon function and sequence homology: pro-survival members include Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w; pro-apoptotic proteins include Bax, Bak and Bok; and "BH3 only" proteins Bad, Bik, Bid, Puma, Bim, Bmf, Noxa and Hrk. Interactions between death-promoting and death-suppressing Bcl-2 family members has led to a rheostat model in which the ratio of pro-apoptotic and anti-apoptotic proteins controls cell fate (4). Thus, pro-survival members exert their behavior by binding to and antagonizing death-promoting members. In general, the "BH3-only members" can bind to and antagonize the pro-survival proteins leading to increased apoptosis (5). While some redundancy of this system likely exists, tissue specificity, transcriptional and post-translational regulation of many of these family members can account for distinct physiological roles.
Like other family members Bok was identified based on sequence homology and interaction with select anti-apoptotic family members (6,7). Although Bok was originally described to be predominantly expressed in reproductive tissues such as ovary, testis and uterus (6), subsequent studies have found selective expression of Bok in a number of adult and development tissues (7). The Bok gene is frequently mutated in human tumors, suggesting a tumor suppressor activity (8). While Bok is structurally similar to the pro-apoptotic proteins Bax and Bak, it appears to be functionally distinct. Loss of Bok in mice did not have an overt effects phenotype (9). However, additional studies have shown that Bok plays a distinct role in apoptosis in response to endoplasmic reticulum stress (10,11). The stability of Bok is regulated by the endoplasmic-reticulum-associated degradation (ERAD) pathway (11). Bok expression is induced by proteasome inhibitors and triggers apoptosis by these agents (11).
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