Carbonic Anhydrase 3/CA3 Antibody (Rabbit mAb) [N13C2]

Application: Reactivity: Mouse, Rat, Human

Lane 1: Rat muscle, Lane 2: Mouse muscle

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 - 1:5000 IHC1:50 - 1:100

Application
WB, 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
30 kDa

Positive Control	Human skeletal muscle tissues; Rat muscle tissues; Mouse muscle tissues
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, 60 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 1:1000), 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, 60 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 1:1000), 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.

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
Carbonic Anhydrase 3/CA3 Antibody (Rabbit mAb) [N13C2] detects endogenous levels of total Carbonic Anhydrase 3/CA3 protein.

Subcellular Location
Cytoplasm

Uniprot ID
P07451

Clone
N13C2

Synonym(s)
Carbonic anhydrase 3 \| Carbonate dehydratase III \| Carbonic anhydrase III \| CA-III \| CA3

Background
Carbonic anhydrase 3 (CA3) is a cytosolic α‑carbonic anhydrase isozyme encoded by the CA3 gene and enriched in mesoderm-derived tissues, particularly skeletal muscle, liver and adipocytes, where it catalyzes the reversible hydration of carbon dioxide but also performs specialized roles in pH regulation, oxidative stress handling and tissue remodeling. The protein shares the conserved zinc metalloenzyme fold of α‑CAs, with a zinc ion coordinated in the active site and residues forming a proton-transfer network, yet it displays markedly lower catalytic activity than ubiquitous isoforms such as CA II, in part because a phenylalanine at position 198 creates steric constriction near the zinc-bound solvent and alters interactions needed for efficient proton transfer. Substitution of Phe198 with leucine increases activity toward CO2 hydration, and exogenous imidazole-containing proton donors can rescue catalytic efficiency, indicating that CA3’s active-site architecture is tuned away from maximal hydration turnover and that proton transfer sites closer to the zinc are critical modulators of its enzymatic profile. Beyond catalysis, CA3 contains surface-exposed cysteine residues that undergo glutathionylation and other redox modifications under oxidative stress, and CA3 overexpression modulates redox-sensitive signaling and protects muscle cells from stress, suggesting a role as a redox sensor and buffer rather than solely as a CO2 hydrase. CA3 is a mesodermal marker: its mRNA is present in primitive mesoderm before myogenesis and later defines subsets of mesodermal cell types, including slow-twitch skeletal muscle fibers, notochord and adipocytes, with expression patterns consistent with regulation by myogenic determination factors and roles in facilitated CO2 diffusion and processes involving proton and bicarbonate transport during muscle function. In mouse skeletal muscle, CA3 contributes to fatigue resistance by supporting intracellular pH homeostasis; muscles lacking CA3 display lower intracellular pH under fatigue conditions, and transgenic expression in cardiomyocytes enhances tolerance to acidosis, maintaining ventricular pressure, systolic and diastolic velocities, and stroke volume under low pH stress, supporting a broader role for CA3 in muscle and cardiac adaptation to metabolic acidosis. In the context of cardiac injury, CA3 is required for appropriate fibrosis and repair after myocardial infarction via regulation of Smad7–Smad2/3 signaling in cardiac fibroblasts, with CA3 deficiency leading to impaired fibrotic responses and adverse remodeling, linking its CO2/pH regulatory function and redox activity to TGF‑β/Smad pathway control during tissue repair. In oral squamous cell carcinoma, CA3 overexpression promotes cell migration and epithelial–mesenchymal transition, with associated changes in E‑cadherin and EMT markers.

Background

Carbonic anhydrase 3 (CA3) is a cytosolic α‑carbonic anhydrase isozyme encoded by the CA3 gene and enriched in mesoderm-derived tissues, particularly skeletal muscle, liver and adipocytes, where it catalyzes the reversible hydration of carbon dioxide but also performs specialized roles in pH regulation, oxidative stress handling and tissue remodeling. The protein shares the conserved zinc metalloenzyme fold of α‑CAs, with a zinc ion coordinated in the active site and residues forming a proton-transfer network, yet it displays markedly lower catalytic activity than ubiquitous isoforms such as CA II, in part because a phenylalanine at position 198 creates steric constriction near the zinc-bound solvent and alters interactions needed for efficient proton transfer. Substitution of Phe198 with leucine increases activity toward CO2 hydration, and exogenous imidazole-containing proton donors can rescue catalytic efficiency, indicating that CA3’s active-site architecture is tuned away from maximal hydration turnover and that proton transfer sites closer to the zinc are critical modulators of its enzymatic profile. Beyond catalysis, CA3 contains surface-exposed cysteine residues that undergo glutathionylation and other redox modifications under oxidative stress, and CA3 overexpression modulates redox-sensitive signaling and protects muscle cells from stress, suggesting a role as a redox sensor and buffer rather than solely as a CO2 hydrase. CA3 is a mesodermal marker: its mRNA is present in primitive mesoderm before myogenesis and later defines subsets of mesodermal cell types, including slow-twitch skeletal muscle fibers, notochord and adipocytes, with expression patterns consistent with regulation by myogenic determination factors and roles in facilitated CO2 diffusion and processes involving proton and bicarbonate transport during muscle function. In mouse skeletal muscle, CA3 contributes to fatigue resistance by supporting intracellular pH homeostasis; muscles lacking CA3 display lower intracellular pH under fatigue conditions, and transgenic expression in cardiomyocytes enhances tolerance to acidosis, maintaining ventricular pressure, systolic and diastolic velocities, and stroke volume under low pH stress, supporting a broader role for CA3 in muscle and cardiac adaptation to metabolic acidosis. In the context of cardiac injury, CA3 is required for appropriate fibrosis and repair after myocardial infarction via regulation of Smad7–Smad2/3 signaling in cardiac fibroblasts, with CA3 deficiency leading to impaired fibrotic responses and adverse remodeling, linking its CO2/pH regulatory function and redox activity to TGF‑β/Smad pathway control during tissue repair. In oral squamous cell carcinoma, CA3 overexpression promotes cell migration and epithelial–mesenchymal transition, with associated changes in E‑cadherin and EMT markers.

References
https://pubmed.ncbi.nlm.nih.gov/1341041/ https://pubmed.ncbi.nlm.nih.gov/22803676/

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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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%