|H M R Mk||Endogenous||290||Rabbit IgG|
Western blot analysis of extracts from various cell lines using SETD2 (D5T1Q) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, SKRC-7 cells are negative for SETD2 expression.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: 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
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.
SETD2 (D5T1Q) Rabbit mAb recognizes endogenous levels of total SETD2 protein.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly1294 of human SETD2 protein.
SET domain-containing protein 2 (SETD2 or SET2), also known as lysine N-methyltransferase 3 A (KMT3A), huntingtin yeast partner B (HYBP), and huntingtin-interacting factor (HIF-1), is a ubiquitously expressed nuclear protein methyltransferase that is responsible for the majority of tri-methylation of histone H3 on lysine 36 (H3K36Me3) (1-3). SETD2-mediated H3K36Me3 is critical for proper regulation of transcription elongation, RNA splicing and DNA mismatch repair (1). SETD2 interacts with RNA polymerase II (RNAPII) that is hyper-phosphorylated on the C-terminal domain (CTD) of the largest subunit Rpb1 (2-4). Upon hyper-phosphorylation of the RNAPII CTD during activation of transcriptional elongation, SETD2 is recruited and facilitates tri-methylation of histone H3 lysine 36 in the body of transcriptionally active genes (2-4). H3K36Me3 then acts to recruit the transcription elongation factor FACT, which modulates nucleosome dynamics to facilitate transcription elongation and prevent cryptic transcriptional initiation (5). In addition, H3K36Me3 acts to recruit RNA-splicing proteins and regulate proper splicing of introns concurrent with transcriptional elongation (3, 6-9). In addition to regulating transcription, SETD2-dependent H3K36Me3 regulates DNA mismatch repair by recruiting MutSα (MSH2 and MSH6) to chromatin during G1 and early S phase (10). Loss of SETD2 results in an increase in microsatellite instability and elevated levels of spontaneous mutations (10). SETD2 is often mutated and/or inactivated in multiple types of cancer, including renal cell carcinoma, leukemia, melanoma and liver cancer (11-13).
Cell Signaling Technology 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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|23486S||100 µl (10 western blots)||$ 255.0|