Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (HRP Conjugate)
What is a Recombinant Antibody and Why is it Important?
Recombinant antibodies offer several key advantages compared to traditional antibodies.
These include superior lot-to-lot consistency, continuous supply, and animal-free manufacturing.
As such, recombinant antibodies are seeing increased use for scientific research, especially as a means of
addressing the ongoing reproducibility crisis.
What is a Recombinant Antibody?
Traditional polyclonal and monoclonal antibodies are the product of normal B cell development and genetic recombination.
They are generated by immunizing an animal with an antigen to elicit an immune response. While polyclonal antibodies are
secreted by many different B cell clones and recognize multiple antigenic epitopes, monoclonals originate from a single B
cell clone and are specific for just one epitope.
Recombinant antibodies are monoclonal, but their production involves in vitro genetic manipulation.
After cloning the antibody genes into an expression vector, this is then transfected into an appropriate host cell line
for antibody expression. Mammalian cell lines are most commonly used for recombinant antibody production, although cell
lines of bacterial, yeast, or insect origin are also suitable.
Superior Lot-to-Lot Consistency
Because recombinant antibody production involves sequencing the antibody light and heavy chains, it is a highly controlled
and reliable process. In contrast, hybridoma-based systems for producing monoclonal antibodies are subject to genetic
drift and instability, increasing the potential for lot-to-lot variability or loss of antibody expression. Recombinant
antibodies are highly consistent from lot to lot, thereby ensuring reproducible experimental results.
In vitro methods for producing antibodies are amenable to large-scale production, meaning antibody availability is
unlikely to become a limiting factor. Moreover, since the recombinant antibody sequence is known, continuity of supply
is assured; in situations where an antibody will be used to support large, long-term studies, this can be an especially
Unlike traditional methods for antibody production, recombinant approaches avoid the need to use animals.
Where polyclonal antibodies are purified directly from the serum of the immunized host, and monoclonals are purified
from either hybridoma-derived tissue culture supernatant or ascites, recombinant antibodies are instead purified from
the tissue culture supernatants of transfected host cell lines.
Regardless of whether an antibody is polyclonal, monoclonal or recombinant, it must always be properly validated
in the intended application prior to experimental use. At CST, we adhere to the
Hallmarks of Antibody Validation™,
six complementary strategies for determining the specificity, sensitivity, and functionality of an antibody in any
given assay. By carefully tailoring these strategies to each antibody product, we guarantee that CST antibodies
will work as expected, to help you achieve results you can trust.
Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (HRP Conjugate) #14056
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 Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173.
Product Usage Information
Supplied in 136 mM NaCl, 2.6 mM KCl, 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 product webpage for recommended antibody dilution.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate.
Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
Microcentrifuge for 5 min.
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.
Electrotransfer to nitrocellulose membrane (#12369).
C. Membrane Blocking and Antibody Incubations
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
I. Membrane Blocking
(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
II. Primary Antibody Incubation
For HRP Conjugated Primary Antibodies
Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
Wash three times for 5 min each with 15 ml of TBST.
Incubate with Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000), to detect biotinylated protein markers, in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
Proceed with detection (Section D).
D. Detection of Proteins
Directions for Use:
Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 264
Specificity / Sensitivity
Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb (HRP Conjugate) recognizes endogenous levels of histone H3 protein only when acetylated at Lys27. This antibody does not cross react with histone H3 acetylated at Lys9, 14, 18, 23, or 56.
Human, Mouse, Rat, Monkey
Species predicted to react based on 100% sequence homology:
The antigen sequence used to produce this antibody shares
100% sequence homology with the species listed here, but
reactivity has not been tested or confirmed to work by CST.
Use of this product with these species is not covered under
Antibody Performance Guarantee.
Hamster, Xenopus, Zebrafish, Horse, Guinea Pig
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding acetylated Lys27 of human histone H3 protein.
The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1,2). Histone acetylation occurs mainly on the amino-terminal tail domains of histones H2A (Lys5), H2B (Lys5, 12, 15, and 20), H3 (Lys9, 14, 18, 23, 27, 36, and 56), and H4 (Lys5, 8, 12, and 16) and is important for the regulation of histone deposition, transcriptional activation, DNA replication, recombination, and DNA repair (1-3). Hyper-acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone-DNA and nucleosome-nucleosome interactions, thereby destabilizing chromatin structure and increasing the accessibility of DNA to various DNA-binding proteins (4,5). In addition, acetylation of specific lysine residues creates docking sites for a protein module called the bromodomain, which binds to acetylated lysine residues (6). Many transcription and chromatin regulatory proteins contain bromodomains and may be recruited to gene promoters, in part, through binding of acetylated histone tails. Histone acetylation is mediated by histone acetyltransferases (HATs), such as CBP/p300, GCN5L2, PCAF, and Tip60, which are recruited to genes by DNA-bound protein factors to facilitate transcriptional activation (3). Deacetylation, which is mediated by histone deacetylases (HDAC and sirtuin proteins), reverses the effects of acetylation and generally facilitates transcriptional repression (7,8).
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