CAMKIV Antibody (Rabbit mAb) [P4P15]

Application: Reactivity: Human

Lane 1: Jurkat

Experiment Essentials

WB
Recommended WB dilution ratio: 1:100000

Usage Information

Dilution
WB1:100000 IF1:50

Application
WB, IF

Reactivity
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
52 kDa 52 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 brain tissue; HeLa cells; Jurkat 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: 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:100000), 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:100000), 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
CAMKIV Antibody (Rabbit mAb) [P4P15] detects endogenous levels of total CAMKIV protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q16566

Clone
P4P15

Synonym(s)
CAMK, CAMK-GR, CAMKIV, CAMK4, Calcium/calmodulin-dependent protein kinase type IV, CaMK IV, CaM kinase-GR

Background
Calcium/calmodulin-dependent protein kinase IV (CaMKIV/CAMK4) is a serine/threonine protein kinase within the Ca2+/calmodulin-dependent kinase family that operates as a nuclear effector of Ca2+ signaling and regulates transcriptional programs in lymphocytes, neurons and male germ cells through phosphorylation of multiple transcription activators. The kinase contains a catalytic domain, an autoinhibitory Ca2+/calmodulin-binding segment and regulatory regions that allow activation by Ca2+/calmodulin and phosphorylation at a threonine residue in the activation loop by upstream CaMKK, followed by intramolecular autophosphorylation that confers autonomous activity even after Ca2+/calmodulin dissociation. Activated CaMKIV is enriched in the nucleus and its best-characterized substrates include CREB, serum response factor and CREB-binding protein; phosphorylation of CREB on Ser133 by CaMKIV enhances CREB transcriptional activity and provides a mechanistic link between transient Ca2+ elevations and more sustained changes in gene expression required for long-term neuronal plasticity and memory consolidation. CaMKIV associates with protein phosphatase 2A via its Ca2+/calmodulin-binding autoinhibitory domain, and PP2A binding is mutually exclusive with Ca2+/calmodulin binding; PP2A dephosphorylates Thr200 in CaMKIV and terminates its autonomous activity, establishing a sequential activation–inactivation cycle in which Ca2+/calmodulin and CaMKK drive T200 phosphorylation and transcriptional stimulation, followed by PP2A-mediated dephosphorylation that limits signal duration. Importin α transports CaMKIV to the nucleus in a manner that does not depend on kinase activity or Ca2+/calmodulin binding, and nuclear localization supports efficient phosphorylation of transcriptional regulators in CREB-mediated signaling pathways, emphasizing that spatial control of CaMKIV is a key element of its function. In Wnt/β‑catenin signaling, the CaMKIV promoter contains TCF/LEF motifs, and canonical Wnt stimulation via Wnt‑3a or lithium increases CaMKIV mRNA and protein expression and promoter activity in hippocampal neurons, with β‑catenin binding to the CaMKIV promoter; Wnt pathway activation in a double transgenic Alzheimer’s disease mouse model restores CaMKIV expression and improves spatial memory, indicating that CaMKIV is a Wnt target gene linked to neuroprotective effects against amyloid pathology. CaMKIV participates in distinct prosurvival pathways in neurons, where dominant-inhibitory CaMKIV alters survival signaling differently from CaMKII inhibition, reinforcing its specific role in coupling Ca2+ signals to long-term transcriptional responses rather than to immediate cytosolic events. Beyond the nervous system, CaMKIV contributes to immune regulation by controlling transcription in lymphocytes and regulating T-cell maturation, dendritic cell survival and cytokine-related inflammatory responses, and it also influences bone growth, sperm motility, microtubule dynamics, cell-cycle progression and apoptosis, placing the kinase at the center of Ca2+-responsive transcriptional control across multiple tissues. Dysregulated CAMKIV activity or expression is associated with cerebral hypoxia, azoospermia, endometrial and ovarian cancers and systemic lupus, supporting interest in CaMKIV as a therapeutic target where its modular domain structure, Ca2+/calmodulin dependence, CaMKK–PP2A regulatory axis and nuclear localization can be exploited to modulate gene expression programs in disease settings.

Background

Calcium/calmodulin-dependent protein kinase IV (CaMKIV/CAMK4) is a serine/threonine protein kinase within the Ca2+/calmodulin-dependent kinase family that operates as a nuclear effector of Ca2+ signaling and regulates transcriptional programs in lymphocytes, neurons and male germ cells through phosphorylation of multiple transcription activators. The kinase contains a catalytic domain, an autoinhibitory Ca2+/calmodulin-binding segment and regulatory regions that allow activation by Ca2+/calmodulin and phosphorylation at a threonine residue in the activation loop by upstream CaMKK, followed by intramolecular autophosphorylation that confers autonomous activity even after Ca2+/calmodulin dissociation. Activated CaMKIV is enriched in the nucleus and its best-characterized substrates include CREB, serum response factor and CREB-binding protein; phosphorylation of CREB on Ser133 by CaMKIV enhances CREB transcriptional activity and provides a mechanistic link between transient Ca2+ elevations and more sustained changes in gene expression required for long-term neuronal plasticity and memory consolidation. CaMKIV associates with protein phosphatase 2A via its Ca2+/calmodulin-binding autoinhibitory domain, and PP2A binding is mutually exclusive with Ca2+/calmodulin binding; PP2A dephosphorylates Thr200 in CaMKIV and terminates its autonomous activity, establishing a sequential activation–inactivation cycle in which Ca2+/calmodulin and CaMKK drive T200 phosphorylation and transcriptional stimulation, followed by PP2A-mediated dephosphorylation that limits signal duration. Importin α transports CaMKIV to the nucleus in a manner that does not depend on kinase activity or Ca2+/calmodulin binding, and nuclear localization supports efficient phosphorylation of transcriptional regulators in CREB-mediated signaling pathways, emphasizing that spatial control of CaMKIV is a key element of its function. In Wnt/β‑catenin signaling, the CaMKIV promoter contains TCF/LEF motifs, and canonical Wnt stimulation via Wnt‑3a or lithium increases CaMKIV mRNA and protein expression and promoter activity in hippocampal neurons, with β‑catenin binding to the CaMKIV promoter; Wnt pathway activation in a double transgenic Alzheimer’s disease mouse model restores CaMKIV expression and improves spatial memory, indicating that CaMKIV is a Wnt target gene linked to neuroprotective effects against amyloid pathology. CaMKIV participates in distinct prosurvival pathways in neurons, where dominant-inhibitory CaMKIV alters survival signaling differently from CaMKII inhibition, reinforcing its specific role in coupling Ca2+ signals to long-term transcriptional responses rather than to immediate cytosolic events. Beyond the nervous system, CaMKIV contributes to immune regulation by controlling transcription in lymphocytes and regulating T-cell maturation, dendritic cell survival and cytokine-related inflammatory responses, and it also influences bone growth, sperm motility, microtubule dynamics, cell-cycle progression and apoptosis, placing the kinase at the center of Ca2+-responsive transcriptional control across multiple tissues. Dysregulated CAMKIV activity or expression is associated with cerebral hypoxia, azoospermia, endometrial and ovarian cancers and systemic lupus, supporting interest in CaMKIV as a therapeutic target where its modular domain structure, Ca2+/calmodulin dependence, CaMKK–PP2A regulatory axis and nuclear localization can be exploited to modulate gene expression programs in disease settings.

References
https://pubmed.ncbi.nlm.nih.gov/26773169/ https://pubmed.ncbi.nlm.nih.gov/15143065/

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%