Phospho-HDAC4 (Ser 246) + HDAC5 (Ser 259) + HDAC7 (Ser 155) Rabbit mAb

Catalog No.: F3397

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Biological Description

Specificity Phospho-HDAC4 (Ser 246) + HDAC5 (Ser 259) + HDAC7 (Ser 155) Rabbit mAb recognizes endogenous levels of total HDAC4 phosphorylated at Ser 246, HDAC5 phosphorylated at Ser 259, and HDAC7 phosphorylated at Ser 155.
Background Phospho-HDAC4 (Ser 246) + HDAC5 (Ser 259) + HDAC7 (Ser 155) are key phosphorylation sites on class IIa histone deacetylases (HDACs), which regulate gene expression by modulating chromatin structure through histone deacetylation. Phosphorylation at these serine residues is a critical mechanism controlling the subcellular localization and activity of these HDACs. Upon phosphorylation, these sites bind to 14-3-3 proteins, sequestering the HDACs in the cytoplasm and preventing their nuclear entry and transcriptional repression activity. HDAC4 phosphorylation at Ser 246, mediated by kinases like CaMKII and PKD, regulates muscle differentiation and neuronal function. Similarly, HDAC5 phosphorylation at Ser 259, also mediated by CaMKII, influences cardiac hypertrophy. HDAC7 phosphorylation at Ser 155, primarily by PKD, affects immune system regulation and vascular biology. These phosphorylation events integrate cellular signalling with epigenetic regulation, ensuring dynamic control of gene expression during development, differentiation, and stress responses. Dysregulation of these processes is linked to diseases such as cancer, neurodegenerative disorders, and cardiovascular conditions.

Usage Information

Application WB, IP Dilution
WB IP
1:1000 1:30
Reactivity Human, Mouse, Rat
Source Rabbit MW 124 kDa, 140 kDa
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
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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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
1389. 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.

Application Data