REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H Mk | Endogenous | 46, 49 | Rabbit IgG |
Western blot analysis of extracts from PANC-1, A549, and 293T (JMJD6 WT and KO) cell lines using JMJD6 (D3O3N) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human colon carcinoma using JMJD6 (D3O3N) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using JMJD6 (D3O3N) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using JMJD6 (D3O3N) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using JMJD6 (D3O3N) Rabbit mAb (left) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded HCT 116 cell pellet (left, high-expressing) or KYSE-70 cell pellet (right, low-expressing) using JMJD6 (D3O3N) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human gastric carcinoma using JMJD6 (D3O3N) Rabbit mAb.
Learn more about how we get our images.For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 (#13953, 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 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 June 2016
Protocol Id: 263
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunohistochemistry (Paraffin) | 1:3200 |
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.
JMJD6 (D3O3N) Rabbit mAb recognizes endogenous levels of total JMJD6 protein.
Human, Monkey
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human JMJD6 protein.
Jumonji domain-containing protein 6 (JMJD6) is a bifunctional metalloenzyme belonging to the large family of JmjC domain-containing proteins. These proteins are ferrous iron- and 2-oxoglutarate-dependent enzymes (Fe2+/2OG) that catalyze hydroxylation and demethylation reactions on a wide variety of protein and nucleic acid substrates (1). JMJD6 is thought to act as both a lysyl-hydroxylase and an arginine demethylase, although the latter activity remains controversial. Specifically, JMJD6 has been shown to catalyze 5-hydroxylation of Lys15 and Lys276 residues on the protein U2AF2 / U2AF65 in vivo, affecting pre-mRNA splicing activity (2). It has also been reported that JMJD6 hydroxylates the Lys382 residue of p53, preventing acetylation and promoting association of p53 with MDMX, resulting in inhibition of p53 transcriptional activity (3). In addition to hydroxylase activity, JMJD6 also acts as an arginine demethylase by targeting histone H3 at Arg2 (H3R2me) and histone H4 at Arg3 (H4R3me). Unlike other members of the JmjC family, JMJD6 appears to have no lysine demethylase activity (4). Studies have shown that JMJD6 colocalizes with BRD4 at a subset of enhancers to demethylate H3R2me2 repressor marks (5). It has also been reported to demethylate non-histone substrates, such as estrogen receptor (ERα) (6), heat shock protein 70 (HSP70) (7), RNA helicase A (8), and the TRAF6 E3 ubiquitin ligase following activation of toll-like receptors (9).
Although mutations in the sequence of JMJD6 have not been observed in cancer, its overexpression is identified in various cancers and is associated with aggressive disease progression and poor prognosis (10). This holds true for certain types of colon (3), lung (11), and breast cancers (12, 13). Based on these findings, JMJD6 has drawn interest as a potential therapeutic target and biomarker for certain cancer types.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalStain is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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Product # | Size | Price | Inventory |
---|---|---|---|
60602S | 100 µl |
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