Western blot analysis of extracts from MCF7 cells, untreated or treated with Trichostatin A (TSA) #9950 (1 µM, 6 hr), using
Acetylated-Lysine (Ac-K-100) MultiMab™ Rabbit mAb mix (HRP Conjugate).
Signal-to-noise (S/N) values of acetylated versus non-acetylated peptides analyzed using
Acetylated-Lysine (Ac-K-100) MultiMab™ Rabbit mAb mix (HRP Conjugate) and 20X LumiGLO® Reagent and 20X Peroxide #7003.
This Cell Signaling Technology® antibody is conjugated by the covalent reaction of hydrazinonicotinamide-modified antibody with formylbenzamide-modified horseradish peroxidase (HRP). The HRP conjugated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Acetylated-Lysine (Ac-K-100) MultiMab™ Rabbit mAb mix (HRP Conjugate) #9814.
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.
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
Acetylated-Lysine (Ac-K-100) MultiMab™ Rabbit mAb mix (HRP Conjugate) detects proteins post-translationally modified by acetylation on the ε-amine groups of lysine residues. The antibody recognizes acetylated lysine in a wide range of sequence contexts. It has been demonstrated to recognize acetylated histones, p53, CBP, PCAF, and chemically acetylated BSA. The antibody has been shown to react with as little as 0.04 ng of chemically acetylated BSA while not recognizing up to 25 µg of non-acetylated BSA. (U.S. Patent No's.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)
MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. Each antibody in the mix is carefully selected based on motif recognition and performance in multiple assays. Each mix is engineered to yield the broadest possible coverage of the modification being studied while ensuring a high degree of specificity for the modification or motif.
Acetylation of lysine, like phosphorylation of serine, threonine or tyrosine, is an important reversible modification controlling protein activity. The conserved amino-terminal domains of the four core histones (H2A, H2B, H3, and H4) contain lysines that are acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs) (1). Signaling resulting in acetylation/deacetylation of histones, transcription factors, and other proteins affects a diverse array of cellular processes including chromatin structure and gene activity, cell growth, differentiation, and apoptosis (2-6). Recent proteomic surveys suggest that acetylation of lysine residues may be a widespread and important form of posttranslational protein modification that affects thousands of proteins involved in control of cell cycle and metabolism, longevity, actin polymerization, and nuclear transport (7,8). The regulation of protein acetylation status is impaired in cancer and polyglutamine diseases (9), and HDACs have become promising targets for anti-cancer drugs currently in development (10).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MultiMab is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
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 firstname.lastname@example.org.