JMJD2A Antibody [G6B24] | Primary Antibodies

Application: Reactivity: Mouse, Rat, Human

Lane 1: 293T, Lane 2: LNCAP, Lane 3: PC12, Lane 4: 3T3

Experiment Essentials

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

Usage Information

Dilution
WB1:5000 IP1:80 IHC1:250

Application
WB, IP, IHC

Reactivity
Mouse, Rat, Human

Source
Rabbit 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 Observed MW
120 kDa 150 kDa,50 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human fetal kidney tissue; Human colon tissue; Rat colon tissue; HAP1 cells; U2OS cells; PC12 cells; HEK-293 cells; LNCaP cells; HeLa cells; NIH/3T3 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/Nuclear 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/Nuclear 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/Nuclear 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 1:5000), 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/Nuclear 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/Nuclear 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/Nuclear 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 1:5000), 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
JMJD2A Antibody (Rabbit mAb) [G6B24] detects endogenous levels of total JMJD2A protein.

Subcellular Location
Nucleus

Uniprot ID
O75164

Clone
G6B24

Synonym(s)
JHDM3A, JMJD2, JMJD2A, KIAA0677, KDM4A, Jumonji domain-containing protein 2A, [histone H3]-trimethyl-L-lysine(36) demethylase 4A, [histone H3]-trimethyl-L-lysine(9) demethylase 4A

Background
JMJD2A (also known as KDM4A) is a JmjC‑domain lysine demethylase of the KDM4/JMJD2 family that targets transcriptionally important methyl marks on histones H3 and H1 to remodel chromatin in a context‑dependent manner, linking its catalytic and scaffolding activities to transcriptional repression, replication control, and lineage‑specific gene activation. The full-length protein contains an N‑terminal catalytic module composed of the JmjN and Fe(II)/2‑oxoglutarate–dependent JmjC domains, followed by a C‑terminal region with double PHD fingers and tandem Tudor domains that recognize methyl‑lysine marks and help position JMJD2A at specific chromatin sites, allowing coordinated substrate recognition and demethylation. JMJD2A demethylates trimethylated lysines on histone H3 at Lys9 and Lys36 and on histone H1.4 at Lys26, with high catalytic efficiency toward H3K9me3 and H1.4K26me3 and little or no activity on mono‑ or dimethylated forms, generating formaldehyde and succinate as reaction products and directly reversing repressive heterochromatic marks that recruit HP1 proteins. Demethylation of H3K9me3 by JMJD2A displaces HP1 from chromatin and has been associated with opening of compacted chromatin and earlier replication timing within normally late‑replicating, H3K9me3‑rich domains, indicating that JMJD2A not only shapes transcriptional potential but also modulates replication origin usage in silent regions by antagonizing HP1‑dependent chromatin organization. At selected promoters, JMJD2A also acts as a transcriptional corepressor: it associates in vivo with pRb, HDACs, and NCOR1 and is recruited to E2F‑responsive and ASCL2 promoters, where it promotes histone deacetylation and maintains H3K9 methylation, reinforcing a repressed chromatin state that constrains cell‑cycle gene expression and lineage‑specific transcription. Alternative splicing generates an N‑terminally truncated isoform (ΔN‑JMJD2A) that lacks the catalytic JmjC demethylase domain but retains C‑terminal recognition modules; this isoform accumulates during skeletal muscle differentiation, binds the Myog promoter at the onset of myogenesis, and is required for myotube formation and muscle gene expression, where its presence correlates with loss of H3K9me2/3 and transcriptional activation, consistent with a role in directing other demethylases and coactivators to remove repressive marks at myogenic loci. ΔN‑JMJD2A occupancy is enriched at genes involved in transcriptional control, and exon-specific siRNA knockdown targeting this isoform selectively impairs MyoD‑driven conversion of fibroblasts to muscle cells, reinforcing its function as a non‑catalytic, chromatin‑bound coactivator module embedded in the JMJD2A locus. Dysregulation of JMJD2A expression or activity alters global levels of H3K9me3/H3K36me3 and H1.4K26me3 and contributes to aberrant transcriptional programs, DNA replication stress, and genomic instability, consistent with its dual ability to erase heterochromatic marks, redistribute HP1, and partner with HDACs and pRb/NCOR1 complexes on growth‑regulatory promoters.

Background

JMJD2A (also known as KDM4A) is a JmjC‑domain lysine demethylase of the KDM4/JMJD2 family that targets transcriptionally important methyl marks on histones H3 and H1 to remodel chromatin in a context‑dependent manner, linking its catalytic and scaffolding activities to transcriptional repression, replication control, and lineage‑specific gene activation. The full-length protein contains an N‑terminal catalytic module composed of the JmjN and Fe(II)/2‑oxoglutarate–dependent JmjC domains, followed by a C‑terminal region with double PHD fingers and tandem Tudor domains that recognize methyl‑lysine marks and help position JMJD2A at specific chromatin sites, allowing coordinated substrate recognition and demethylation. JMJD2A demethylates trimethylated lysines on histone H3 at Lys9 and Lys36 and on histone H1.4 at Lys26, with high catalytic efficiency toward H3K9me3 and H1.4K26me3 and little or no activity on mono‑ or dimethylated forms, generating formaldehyde and succinate as reaction products and directly reversing repressive heterochromatic marks that recruit HP1 proteins. Demethylation of H3K9me3 by JMJD2A displaces HP1 from chromatin and has been associated with opening of compacted chromatin and earlier replication timing within normally late‑replicating, H3K9me3‑rich domains, indicating that JMJD2A not only shapes transcriptional potential but also modulates replication origin usage in silent regions by antagonizing HP1‑dependent chromatin organization. At selected promoters, JMJD2A also acts as a transcriptional corepressor: it associates in vivo with pRb, HDACs, and NCOR1 and is recruited to E2F‑responsive and ASCL2 promoters, where it promotes histone deacetylation and maintains H3K9 methylation, reinforcing a repressed chromatin state that constrains cell‑cycle gene expression and lineage‑specific transcription. Alternative splicing generates an N‑terminally truncated isoform (ΔN‑JMJD2A) that lacks the catalytic JmjC demethylase domain but retains C‑terminal recognition modules; this isoform accumulates during skeletal muscle differentiation, binds the Myog promoter at the onset of myogenesis, and is required for myotube formation and muscle gene expression, where its presence correlates with loss of H3K9me2/3 and transcriptional activation, consistent with a role in directing other demethylases and coactivators to remove repressive marks at myogenic loci. ΔN‑JMJD2A occupancy is enriched at genes involved in transcriptional control, and exon-specific siRNA knockdown targeting this isoform selectively impairs MyoD‑driven conversion of fibroblasts to muscle cells, reinforcing its function as a non‑catalytic, chromatin‑bound coactivator module embedded in the JMJD2A locus. Dysregulation of JMJD2A expression or activity alters global levels of H3K9me3/H3K36me3 and H1.4K26me3 and contributes to aberrant transcriptional programs, DNA replication stress, and genomic instability, consistent with its dual ability to erase heterochromatic marks, redistribute HP1, and partner with HDACs and pRb/NCOR1 complexes on growth‑regulatory promoters.

References
https://pubmed.ncbi.nlm.nih.gov/23644528/ https://pubmed.ncbi.nlm.nih.gov/21694756/

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%

JMJD2A Antibody (Rabbit mAb) [G6B24]