HDAC5 Antibody [B2J1] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat, Monkey

Lane 1: Saso-2, Lane 2: Hela, Lane 3: T47D, Lane 4: THP-1

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:200 CHIP1:50

Application
WB, IP, ChIP

Reactivity
Human, Mouse, Rat, Monkey

Source
Mouse Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
140 kDa

Positive Control	THP-1 cells; 3T3-L1 undifferentiated cells; COS7 cells; NIH/3T3 cells; KNRK cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
HDAC5 Antibody (Mouse mAb) [B2J1] detects endogenous levels of total HDAC5 protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q9UQL6

Clone
B2J1

Synonym(s)
Histone deacetylase 5, HD5, Antigen NY-CO-9, HDAC5, KIAA0600

Background
HDAC5 is a class IIa histone deacetylase that acts as a signal‑regulated transcriptional corepressor, linking extracellular and intracellular cues to chromatin deacetylation and repression of defined gene programs in muscle, endothelium, heart, brain, and cancer. The protein contains an N‑terminal regulatory region with multiple serine phosphorylation sites, 14‑3‑3‑binding motifs, and interaction regions for transcription factors such as MEF2, GATA1, and SATB1, and a C‑terminal catalytic domain with the conserved HDAC fold that deacetylates lysines in the N‑terminal tails of core histones when assembled into multiprotein complexes with HDAC3 and NCOR/SMRT. HDAC5 tethered to MEF2 or other transcription factors at target promoters maintains low histone acetylation, a compact chromatin state, and reduced transcription of genes involved in myogenesis, angiogenesis, inflammatory responses, and oncogenic programs, and changes in its localization or modification status rapidly alter the expression of these sets. Phosphorylation of HDAC5 at key serines by kinases such as CaMK, PKD, AMPK, SIK1, and GRK5 creates high‑affinity docking sites for 14‑3‑3 proteins and triggers CRM1‑dependent nuclear export, relieving repression of MEF2‑dependent and other target genes and allowing activation of muscle differentiation and hypertrophic gene expression in response to calcium, adrenergic, and metabolic signals. Dephosphorylation by PP2A–B55α complexes or impaired phosphorylation promotes nuclear retention and sustained repression, and these opposing phosphorylation–dephosphorylation cycles integrate neurohormonal and mechanical inputs into dynamic control of HDAC5 nuclear occupancy in cardiomyocytes and vascular cells. In endothelial cells, HDAC5 is a key determinant of the angiogenic gene expression profile: nuclear HDAC5 represses pro‑angiogenic genes, whereas VEGF and shear‑stress–induced export of HDAC5 enhances MEF2‑driven transcription, increases expression of angiogenic mediators, and promotes sprouting and tube formation. In cancer, HDAC5 is frequently dysregulated, and altered HDAC5 levels or localization affect proliferation, migration, epithelial–mesenchymal transition, cancer stemness, and drug sensitivity through deacetylation of chromatin and non‑histone substrates including RARA and SATB1.

Background

HDAC5 is a class IIa histone deacetylase that acts as a signal‑regulated transcriptional corepressor, linking extracellular and intracellular cues to chromatin deacetylation and repression of defined gene programs in muscle, endothelium, heart, brain, and cancer. The protein contains an N‑terminal regulatory region with multiple serine phosphorylation sites, 14‑3‑3‑binding motifs, and interaction regions for transcription factors such as MEF2, GATA1, and SATB1, and a C‑terminal catalytic domain with the conserved HDAC fold that deacetylates lysines in the N‑terminal tails of core histones when assembled into multiprotein complexes with HDAC3 and NCOR/SMRT. HDAC5 tethered to MEF2 or other transcription factors at target promoters maintains low histone acetylation, a compact chromatin state, and reduced transcription of genes involved in myogenesis, angiogenesis, inflammatory responses, and oncogenic programs, and changes in its localization or modification status rapidly alter the expression of these sets. Phosphorylation of HDAC5 at key serines by kinases such as CaMK, PKD, AMPK, SIK1, and GRK5 creates high‑affinity docking sites for 14‑3‑3 proteins and triggers CRM1‑dependent nuclear export, relieving repression of MEF2‑dependent and other target genes and allowing activation of muscle differentiation and hypertrophic gene expression in response to calcium, adrenergic, and metabolic signals. Dephosphorylation by PP2A–B55α complexes or impaired phosphorylation promotes nuclear retention and sustained repression, and these opposing phosphorylation–dephosphorylation cycles integrate neurohormonal and mechanical inputs into dynamic control of HDAC5 nuclear occupancy in cardiomyocytes and vascular cells. In endothelial cells, HDAC5 is a key determinant of the angiogenic gene expression profile: nuclear HDAC5 represses pro‑angiogenic genes, whereas VEGF and shear‑stress–induced export of HDAC5 enhances MEF2‑driven transcription, increases expression of angiogenic mediators, and promotes sprouting and tube formation. In cancer, HDAC5 is frequently dysregulated, and altered HDAC5 levels or localization affect proliferation, migration, epithelial–mesenchymal transition, cancer stemness, and drug sensitivity through deacetylation of chromatin and non‑histone substrates including RARA and SATB1.

References
https://pubmed.ncbi.nlm.nih.gov/34178647/ https://pubmed.ncbi.nlm.nih.gov/10869435/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

HDAC5 Antibody (Mouse mAb) [B2J1]