SLC22A3/OCT3 Antibody (Rabbit mAb) [J20M4]

Application: Reactivity: Human

Lane 1: 293T, Lane 2: 293T (SLC22A3 transfected)

Experiment Essentials

WB
Recommended WB dilution ratio: 1:10000

Usage Information

Dilution
WB1:10000 - 1:50000 IHC1:100 - 1:250 FCM1:100

Application
WB, IHC, FCM

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
61 kDa 80 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 muscle tissue; Human placenta tissue; JEG3 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 1:10000), 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 1:10000), 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
SLC22A3/OCT3 Antibody (Rabbit mAb) [J20M4] detects endogenous levels of total SLC22A3/OCT3 protein.

Subcellular Location
Cell membrane, Membrane, Mitochondrion, Nucleus

Uniprot ID
O75751

Clone
J20M4

Synonym(s)
EMTH, OCT3, SLC22A3, Solute carrier family 22 member 3, Extraneuronal monoamine transporter, Organic cation transporter 3, EMT

Background
SLC22A3, also termed OCT3, is a widely expressed polyspecific organic cation transporter of the SLC22 family that mediates low-affinity, high-capacity uptake of endogenous monoamines and a range of cationic drugs, thereby contributing to neurotransmitter clearance, drug disposition, and tissue exposure in organs such as brain, heart, and liver. The protein is a typical solute carrier with multiple transmembrane helices forming a central aqueous pore that alternates access across the plasma membrane in an electrogenic, membrane potential–dependent manner, allowing facilitated diffusion of positively charged substrates including norepinephrine, epinephrine, dopamine, histamine, and neuromodulators such as agmatine and salsolinol, as well as drugs like metformin, lidocaine, quinidine, oxaliplatin, and lamivudine. OCT3 acts as a major extraneuronal monoamine transporter in the central nervous system, where its broad substrate selectivity and high capacity regulate interstitial levels of norepinephrine, serotonin, and other cationic signaling molecules, shaping the spillover of monoamines from synapses, the duration of receptor activation, and behavioral responses. OCT3 activity is linked to susceptibility to depression and other neuropsychiatric conditions, and several clinically used antidepressants inhibit OCT3-mediated uptake in vitro at micromolar concentrations, indicating that OCT3 can serve as an additional target influencing monoaminergic tone beyond high-affinity SLC6 transporters. Expression outside the nervous system extends OCT3 function to cardiovascular and metabolic contexts: strong expression in the human heart, particularly in vascular endothelial cells, supports uptake of catecholamines and cationic drugs into myocardium and contributes to local catecholamine clearance and cardiac drug handling. OCT3-dependent metformin transport into cardiac tissue is inhibited by commonly used cardiovascular drugs such as verapamil and carvedilol, highlighting a transporter-mediated mechanism for drug–drug interactions and potential modification of cardiac drug exposure. SLC22A3 variants are implicated in coronary artery disease and related cardiovascular phenotypes, and specific polymorphisms alter transporter expression, substrate affinity, or uptake capacity, which can change clearance of endogenous cations and OCT3-substrate drugs and thereby modify inflammatory responses, blood pressure regulation, or susceptibility to vascular disease. Across tissues, OCT3 is regulated post-transcriptionally and by signaling pathways such as Ca²⁺/calmodulin, which can acutely enhance cation uptake, providing a mechanism for dynamic adjustment of transporter activity in response to cellular state. These properties define SLC22A3/OCT3 as a structurally conserved, electrogenic organic cation transporter that links monoamine and drug handling in brain, heart, liver, and other organs to neuropsychiatric outcomes, cardiovascular risk, and variability in therapeutic response, making it a key target for studies on monoaminergic signaling, cardiotoxicity mitigation, and pharmacogenetic optimization of cationic drugs.

Background

SLC22A3, also termed OCT3, is a widely expressed polyspecific organic cation transporter of the SLC22 family that mediates low-affinity, high-capacity uptake of endogenous monoamines and a range of cationic drugs, thereby contributing to neurotransmitter clearance, drug disposition, and tissue exposure in organs such as brain, heart, and liver. The protein is a typical solute carrier with multiple transmembrane helices forming a central aqueous pore that alternates access across the plasma membrane in an electrogenic, membrane potential–dependent manner, allowing facilitated diffusion of positively charged substrates including norepinephrine, epinephrine, dopamine, histamine, and neuromodulators such as agmatine and salsolinol, as well as drugs like metformin, lidocaine, quinidine, oxaliplatin, and lamivudine. OCT3 acts as a major extraneuronal monoamine transporter in the central nervous system, where its broad substrate selectivity and high capacity regulate interstitial levels of norepinephrine, serotonin, and other cationic signaling molecules, shaping the spillover of monoamines from synapses, the duration of receptor activation, and behavioral responses. OCT3 activity is linked to susceptibility to depression and other neuropsychiatric conditions, and several clinically used antidepressants inhibit OCT3-mediated uptake in vitro at micromolar concentrations, indicating that OCT3 can serve as an additional target influencing monoaminergic tone beyond high-affinity SLC6 transporters. Expression outside the nervous system extends OCT3 function to cardiovascular and metabolic contexts: strong expression in the human heart, particularly in vascular endothelial cells, supports uptake of catecholamines and cationic drugs into myocardium and contributes to local catecholamine clearance and cardiac drug handling. OCT3-dependent metformin transport into cardiac tissue is inhibited by commonly used cardiovascular drugs such as verapamil and carvedilol, highlighting a transporter-mediated mechanism for drug–drug interactions and potential modification of cardiac drug exposure. SLC22A3 variants are implicated in coronary artery disease and related cardiovascular phenotypes, and specific polymorphisms alter transporter expression, substrate affinity, or uptake capacity, which can change clearance of endogenous cations and OCT3-substrate drugs and thereby modify inflammatory responses, blood pressure regulation, or susceptibility to vascular disease. Across tissues, OCT3 is regulated post-transcriptionally and by signaling pathways such as Ca²⁺/calmodulin, which can acutely enhance cation uptake, providing a mechanism for dynamic adjustment of transporter activity in response to cellular state. These properties define SLC22A3/OCT3 as a structurally conserved, electrogenic organic cation transporter that links monoamine and drug handling in brain, heart, liver, and other organs to neuropsychiatric outcomes, cardiovascular risk, and variability in therapeutic response, making it a key target for studies on monoaminergic signaling, cardiotoxicity mitigation, and pharmacogenetic optimization of cationic drugs.

References
https://pubmed.ncbi.nlm.nih.gov/21697722/ https://pubmed.ncbi.nlm.nih.gov/22342816/

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%