|H M R Mk All||Endogenous||Mouse IgM|
Western blot analysis of extracts from HeLa cells, untreated (-) or treated with PUGNAc (+), an inhibitor of N-acetyl-β-D-glucosaminidase, using O-GlcNAc (CTD110.6) Mouse mAb (HRP Conjugate).Learn more about how we get our images.
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.
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: 264
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
O-GlcNAc (CTD110.6) Mouse mAb (HRP Conjugate) specifically recognizes endogenous levels of O-GlcNAc on proteins in β-O-glycosidic linkage to both serine and threonine.
Human, Mouse, Rat, Monkey, All Species Expected
Monoclonal antibody is produced by immunizing animals with a peptide containing serine-O-linked N-acetylglucosamine (O-GlcNAc).
This Cell Signaling Technology® antibody is conjugated to the carbohydrate groups of horseradish peroxidase (HRP) via its amine groups. The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated O-GlcNAc (CTD110.6) Mouse mAb #9875.
A distinct form of protein glycosylation, beta-linked N-acetyl-glucosamine (GlcNAc) moieties can be added to Serine or Threonine residues of proteins. (1,2) This differs from other forms of glycosylation, as it typically is a single moiety rather than the complex branched sugars that are more commonly studied. It is thought that these modifications happen in a much more dynamic cycle more reminiscent of phosphorylation modifications. (4) GlcNAc modified proteins are found in the cytoplasm and nucleus and are modulated by means of specific O-GlcNAc transferases (OGT) as well as GlcNAcase activity that can be inhibited using the Thiamet-G (TMG) inhibitor. Mass spectrometry analysis of this modification has been complicated due to the loss of the GlcNAc group during ionization and fragmentation but methods and technologies such as electron transfer dissociation (ETD) are opening up new avenues to study these modifications. Indications are that O-GlcNAc could play important roles in many cellular processes including metabolism, growth, morphogenesis, apoptosis, transcription and may play a critical role in cancer.(3)
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.
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|12938S||100 µl (10 western blots)||$ 297.0|