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 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 June 2020
Protocol Id: 10
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human ALKBH1 protein.
AlkB is an oxidative dealkylating DNA repair enzyme first characterized in E.coli (1-5). Nine AlkB homologs exist in mammals, with the first eight designated as ALKBH1-ALKBH8, and the ninth as FTO (fat mass and obesity-associated protein) (6). ALKBH1, which features the highest sequence identity to E.coli AlkB, is an Fe(II) and 2-oxoglutarate-dependent dioxygenase that acts upon nucleic acids such as DNA and tRNA and carries out a wide range of enzymatic functions (6,7). Similar to other AlkB proteins, ALKBH1 is able to repair alkylated single-stranded DNA and RNA containing 3-methylcytosine (m3C), albeit with weak activity (8). Perhaps more importantly, it has also been shown to catalyze the demethylation of N1-methyladenosine on tRNAs to regulate translation (9). ALKBH1 functions in the mitochondria as well, recognizing and oxidizing 5-methylcytosine (m5C) on mitochondrial tRNAMet to generate 5-formylcytosine, consequently enhancing mitochondrial translation (10). Interestingly, ALKBH1 has also been shown to possess apurinic/apyrimidinic (AP) lyase activity, cleaving both single-stranded and double-stranded DNA at abasic sites, with greatest affinity toward double-stranded DNA with two abasic sites (11). Lastly, ALKBH1 has been reported to possess N(6)-methyladenine (6mA) demethylase activity, suggesting a role in epigenetic regulation (12,13). However, an additional study was unable to show definitive ALKBH1 6mA demethylase activity using both biochemistry assays and knockout mice, so this enzymatic function remains controversial (9).
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