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Anti-Histone H3 (Trimethyl Lys4, Serotonyl Gln5) Rabbit pAb

Catalog Number: PTM-1420

$ 390

Clone Number: /

Host: Rabbit Clonality: Polyclonal

Applications： WB ChIP

Reactivity: Human, Mouse, Rat

Synonyms: H3K4me3/Q5ser

Product Size

100 μl

Quantity

Shipping: Ambient temperature

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General Information
Product Usage Information
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General Information

Isotype	IgG
Conjugate	Unconjugated
Synonyms	H3K4me3/Q5ser
UniProt ID	P68431
Immunogen	Trimethylated/serotonylated human histone H3 (Lys4/Gln5) peptide
MW (kDa)	15
Specificity	Anti-Trimethyl/Serotonyl-Histone H3 (Lys4/Gln5) Rabbit pAb detects histone H3 only when it is trimethylated at Lys4 and serotonylated at Gln5.

Product Usage Information

Applications	Dilution	Recommended Species
WB	1:500 - 1:2000	Human, Mouse, Rat
ChIP	6 μg per 5x10⁶ cells	Human

Properties

Purity	Protein A and immunogen affinity purified
Constituents	PBS, Glycerol, BSA
Storage	Store at -20°C. Avoid freeze/thaw cycles.
Stability	Stable for 12 months from date of receipt/reconstitution.

Target Information

Background

Histone post-translational modifications (PTMs) are key mechanisms of epigenetics that modulate chromatin structures, termed as “histone code”. Serotonin, also known as 5-hydroxytryptamine (5-HT), is an excitatory neurotransmitter that regulates brain cell activity by activating specific membrane receptors in the brain. The amino group of serotonins can covalently bind to the glutamine residue at the fifth position of histone H3 by TGM2 to form histone 5-hydroxytryptamine (H3Q5ser). H3Q5ser modifies sites adjacent to H3K4me3 form PTM crosstalk. The presence of H3K4me3Q5ser enhanced the binding of transcription factors, such as TFIID to chromatin, thereby activating the expression of downstream genes in developing rodent brains and human neurons.

Cellular location

Nucleus

Images

Dot Blot
	Peptide amount: 1 ng, 4 ng, 16 ng Blocking buffer: 5% NFDM/TBST Primary Ab dilution: 1:2000 Primary Ab incubation: 2 hours at room temperature Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate) Exposure time: 60 seconds The list of peptides used in the experiment is provided below. Lane 1: H3K4 trimethyl, Q5 serotonyl. Lane 2: H3K4 monomethyl. Lane 3: H3K4 dimethyl. Lane 4: H3K4 trimethyl. Lane 5: H3K4 crotonyl. Lane 6: H3Q5 serotonyl. Lane 7: H3K9 trimethyl. Lane 8: H3K14 trimethyl. Lane 9: H3K4/Q5 unmodified.

Dot Blot

Peptide amount: 1 ng, 4 ng, 16 ng
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:2000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 60 seconds
The list of peptides used in the experiment is provided below.
Lane 1: H3K4 trimethyl, Q5 serotonyl. Lane 2: H3K4 monomethyl.
Lane 3: H3K4 dimethyl. Lane 4: H3K4 trimethyl.
Lane 5: H3K4 crotonyl. Lane 6: H3Q5 serotonyl.
Lane 7: H3K9 trimethyl. Lane 8: H3K14 trimethyl.
Lane 9: H3K4/Q5 unmodified.

WB
	Lysates: HeLa, N2a, BRL, mouse kidney Protein loading amount: 20 μg Blocking buffer: 5% NFDM/TBST Primary Ab dilution: 1:2000 Primary Ab incubation: 2 hours at room temperature Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate) Exposure time: 60 seconds Predicted band size: 15 kDa Observed band size: 15 kDa
	Lysates: N2a cells Protein loading amount: 20 μg Blocking buffer: 5% NFDM/TBST Primary Ab: 1. PTM-1420 pre-adsorbed with synthetic H3K4me3/Q5ser peptides; 2. PTM-1420 pre-adsorbed with synthetic unmodified peptides; 3. PTM-1420 with no peptide blocking Primary Ab dilution: 1:2000 Primary Ab incubation: 2 hours at room temperature Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate) Exposure time: 60 seconds Predicted band size: 15 kDa Observed band size: 15 kDa

Lysates: HeLa, N2a, BRL, mouse kidney
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab dilution: 1:2000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 60 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

Lysates: N2a cells
Protein loading amount: 20 μg
Blocking buffer: 5% NFDM/TBST
Primary Ab: 1. PTM-1420 pre-adsorbed with synthetic H3K4me3/Q5ser peptides; 2. PTM-1420 pre-adsorbed with synthetic unmodified peptides; 3. PTM-1420 with no peptide blocking
Primary Ab dilution: 1:2000
Primary Ab incubation: 2 hours at room temperature
Secondary Ab: Goat Anti-Rabbit IgG H&L pAb (HRP Conjugate)
Exposure time: 60 seconds
Predicted band size: 15 kDa
Observed band size: 15 kDa

ChIP
	Sample: HeLa cells treated with 100 mM sodium lactate for 24 hours Cross-linking conditions: no cross-linking Amount of chromatin per IP: 5x10⁶ cells Amount of Ab per IP: 6 μg Beads type and amount per IP: 50 μL of Protein A MagBeads Description: Chromatin immunoprecipitations were performed with 6 μg of normal rabbit IgG as a negative control. The immunoprecipitated DNA was quantified by real-time PCR using primers specific for the human GAPDH-CDS, RPL30 excon 3, LDHA, FOXO3a-promoter, FOXO3a-downstream, RAB20, and LDHA-promoter regions. The data are presented as enrichment of each sample relative to total amount of input at each amplicon.

ChIP

Sample: HeLa cells treated with 100 mM sodium lactate for 24 hours
Cross-linking conditions: no cross-linking
Amount of chromatin per IP: 5x10⁶ cells
Amount of Ab per IP: 6 μg
Beads type and amount per IP: 50 μL of Protein A MagBeads
Description: Chromatin immunoprecipitations were performed with 6 μg of normal rabbit IgG as a negative control. The immunoprecipitated DNA was quantified by real-time PCR using primers specific for the human GAPDH-CDS, RPL30 excon 3, LDHA, FOXO3a-promoter, FOXO3a-downstream, RAB20, and LDHA-promoter regions. The data are presented as enrichment of each sample relative to total amount of input at each amplicon.

Research Use

For research use only, not for use in diagnostic procedures.