Western blot analysis of extracts from U266, K-562, and Raji cells using ALKBH7 Antibody.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length Myc/DDK-tagged human ALKBH7 protein (hALKBH7-Myc/DDK; +), using ALKBH7 Antibody (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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
ALKBH7 Antibody recognizes endogenous levels of total ALKBH7 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val34 of human ALKBH7 protein. Antibodies are purified by protein A and peptide affinity chromatography.
The AlkB alkylation repair homolog 7 (ALKBH7, ABH7) is a member of the alkylated DNA repair homolog family that is responsible for repair of DNA damage induced by oxidation and alkylation (1). ALKBH7 is a nuclear encoded protein that contains an amino-terminal mitochondrial targeting sequence that directs import of ALKBH7 to the mitochondria (2). Unlike other Alkb family members, the ALKBH7 protein lacks a functional nucleotide recognition lid essential for nucleobase-binding, which abrogates any DNA or RNA repair capability (3). In response to DNA damage, mitochondrial ALKBH7 triggers the collapse of the mitochondrial membrane potential. The resultant loss of mitochondrial function leads to depletion of cellular energy and programmed cell death (2). Research studies indicate that ALKBH7 knockdown cells are resistant to apoptotic cell death induced by oxidizing and alkylating agents, which suggests that ALKBH7 may play a novel function in promoting cell death (2). Indeed, ALKBH7 has been identified as a key regulator of the alkylation or oxidizing DNA damaged induced necroptosis pathway (2).
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