Cat. # | Size | Qty. | Price |
---|---|---|---|
39013S | 100 µl |
|
REACTIVITY | H Mk |
SENSITIVITY | Endogenous |
MW (kDa) | 44 |
SOURCE | Rabbit |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
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: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
Proceed to one of the following specific set of steps.
NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2008
revised October 2021
Protocol Id: 409
Human, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ALKBH1 protein. Antibodies are purified by peptide affinity chromatography.
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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