PDE4B Antibody [A23H17] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: A-204, Lane 2: Mouse brain, Lane 3: Rat brain

Usage Information

Dilution
WB1:1000 IP1:150

Application
WB, IP

Reactivity
Human, Mouse, Rat

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
90 kDa

Positive Control	Mouse brain; Rat brain
Negative Control	A-204 cells

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: 10%. 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: 10%. 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
PDE4B Antibody (Rabbit mAb) [A23H17] detects endogenous levels of total PDE4B protein.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
Q07343

Clone
A23H17

Synonym(s)
3',5'-cyclic-AMP phosphodiesterase 4B, DPDE4, PDE32, cAMP-specific phosphodiesterase 4B, PDE4B, DPDE4

Background
PDE4B is a cAMP‑specific phosphodiesterase of the PDE4 family that hydrolyzes 3′,5′‑cAMP to AMP and sets local cAMP levels in multiple immune and neural cell types, placing it as a key negative regulator of cAMP‑dependent signaling. The protein contains the conserved catalytic core of class I PDEs, with metal‑binding His and Asp residues forming the active site that coordinates divalent cations and positions cAMP for nucleophilic attack, and N‑terminal regulatory regions that target PDE4B isoforms to defined subcellular compartments and scaffold complexes. In dendritic cells and monocytes, PDE4B is a predominant cAMP‑degrading enzyme, and its activity limits cAMP accumulation after receptor stimulation, thereby controlling the amplitude and duration of PKA‑ and EPAC‑dependent phosphorylation events that influence NF‑κB, CREB, and other transcription factors. By shaping these cAMP gradients, PDE4B regulates production of inflammatory cytokines such as TNF‑α, IL‑6, and IL‑1β, affects expression of costimulatory molecules, and modulates the balance between pro‑ and anti‑inflammatory programs during responses to bacterial lipopolysaccharide and other stimuli. In cardiac myocytes, PDE4B localizes to a sarcolemmal compartment associated with β1‑adrenergic receptors, and its catalytic activity forms part of a local negative feedback loop that constrains cAMP signals generated by β1‑adrenergic stimulation, thereby tuning PKA phosphorylation of nearby substrates that regulate calcium handling and contractility. Genetic deletion or pharmacologic inhibition of PDE4B in this compartment leads to enhanced β1‑adrenergic cAMP responses and altered phosphorylation of proteins linked to excitation–contraction coupling.

Background

PDE4B is a cAMP‑specific phosphodiesterase of the PDE4 family that hydrolyzes 3′,5′‑cAMP to AMP and sets local cAMP levels in multiple immune and neural cell types, placing it as a key negative regulator of cAMP‑dependent signaling. The protein contains the conserved catalytic core of class I PDEs, with metal‑binding His and Asp residues forming the active site that coordinates divalent cations and positions cAMP for nucleophilic attack, and N‑terminal regulatory regions that target PDE4B isoforms to defined subcellular compartments and scaffold complexes. In dendritic cells and monocytes, PDE4B is a predominant cAMP‑degrading enzyme, and its activity limits cAMP accumulation after receptor stimulation, thereby controlling the amplitude and duration of PKA‑ and EPAC‑dependent phosphorylation events that influence NF‑κB, CREB, and other transcription factors. By shaping these cAMP gradients, PDE4B regulates production of inflammatory cytokines such as TNF‑α, IL‑6, and IL‑1β, affects expression of costimulatory molecules, and modulates the balance between pro‑ and anti‑inflammatory programs during responses to bacterial lipopolysaccharide and other stimuli. In cardiac myocytes, PDE4B localizes to a sarcolemmal compartment associated with β1‑adrenergic receptors, and its catalytic activity forms part of a local negative feedback loop that constrains cAMP signals generated by β1‑adrenergic stimulation, thereby tuning PKA phosphorylation of nearby substrates that regulate calcium handling and contractility. Genetic deletion or pharmacologic inhibition of PDE4B in this compartment leads to enhanced β1‑adrenergic cAMP responses and altered phosphorylation of proteins linked to excitation–contraction coupling.

References
https://pubmed.ncbi.nlm.nih.gov/35387344/ https://pubmed.ncbi.nlm.nih.gov/36278172/

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%

PDE4B Antibody (Rabbit mAb) [A23H17]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support