Connexin 36 Antibody [L23J6]

Catalog No.: F3937

Application: Reactivity: Human, Mouse

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution
IHC1:160-1:250 IF1:160-1:250

Application
IHC, IF

Source
Mouse Monoclonal Antibody

Reactivity
Human, Mouse

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

ポジティブコントロール	Adult mouse retina
ネガティブコントロール

プロトコール

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.

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.

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

生物学的記述

Specificity
Connexin 36 Antibody [L23J6] detects endogenous levels of total Connexin 36 protein.

タンパク質の局在
細胞接着、細胞膜、ギャップ結合、細胞内膜系

Uniprot ID
Q9UKL4

Clone
L23J6

Synonym(s)
connexin 36; Connexin-36; Cx36; Gap junction alpha-9 protein; Gap junction delta-2 protein; gap junction membrane channel protein alpha 9; gap junction protein, alpha 9; gap junction protein, delta 2, 36kDa; GJA10; connexin36; CX36; GJA9; GJD2

Background
Connexin 36 (Cx36, GJC1) is a neuron-specific gap junction protein highly expressed in mammalian brain interneurons and retinal neurons, assembling into hexameric connexons where each monomer features four transmembrane domains (TM1–4); TM1 and TM2 form the pore-lining hydrophobic constriction, flanked by extracellular loops (EL1/EL2) containing conserved d-prolines (Pro47, Pro187) that facilitate intercellular docking. The channel possesses a short intracellular N-terminal tail with CaMKII phosphorylation sites (notably Ser293), and a cytoplasmic loop (CL) domain that mediates Ca²⁺/calmodulin binding, allosterically gating the ~15 Å diameter aqueous pore, which is selectively permeable to cations and anions under 1.2 kDa (including K⁺, IP₃, cAMP, and Lucifer yellow). While voltage-insensitive, Cx36 gap junction channels are sensitive to pH and Ca²⁺, exhibit low unitary conductance (~15 pS), and mediate bidirectional electrotonic coupling for precise spikelet synchrony across inhibitory networks by allowing direct current flow between somata and dendrites. CaMKII-dependent phosphorylation at Ser293 increases open probability during synaptic plasticity, whereas pharmacological agents like mefloquine and quinine inhibit channel conductance by binding inter-monomer TM1/TM2 hydrophobic pockets (I35/V38/A39/I40, I76/V80), forming occlusive rings that dehydrate the ion permeation pathway and reduce conductance by over 50%. Cx36 is essential for gamma oscillations, phase-locking in suprachiasmatic nucleus (SCN) circadian pacemakers, and AII amacrine-mediated rod pathway signaling in the retina; its knockout disrupts spike timing precision, impairs visuomotor reflexes, and increases seizure susceptibility. Mutations such as R278H destabilize gap junction plaques, leading to juvenile myoclonic epilepsy and Oculodentodigital dysplasia phenotypes.

Background

Connexin 36 (Cx36, GJC1) is a neuron-specific gap junction protein highly expressed in mammalian brain interneurons and retinal neurons, assembling into hexameric connexons where each monomer features four transmembrane domains (TM1–4); TM1 and TM2 form the pore-lining hydrophobic constriction, flanked by extracellular loops (EL1/EL2) containing conserved d-prolines (Pro47, Pro187) that facilitate intercellular docking. The channel possesses a short intracellular N-terminal tail with CaMKII phosphorylation sites (notably Ser293), and a cytoplasmic loop (CL) domain that mediates Ca²⁺/calmodulin binding, allosterically gating the ~15 Å diameter aqueous pore, which is selectively permeable to cations and anions under 1.2 kDa (including K⁺, IP₃, cAMP, and Lucifer yellow). While voltage-insensitive, Cx36 gap junction channels are sensitive to pH and Ca²⁺, exhibit low unitary conductance (~15 pS), and mediate bidirectional electrotonic coupling for precise spikelet synchrony across inhibitory networks by allowing direct current flow between somata and dendrites. CaMKII-dependent phosphorylation at Ser293 increases open probability during synaptic plasticity, whereas pharmacological agents like mefloquine and quinine inhibit channel conductance by binding inter-monomer TM1/TM2 hydrophobic pockets (I35/V38/A39/I40, I76/V80), forming occlusive rings that dehydrate the ion permeation pathway and reduce conductance by over 50%. Cx36 is essential for gamma oscillations, phase-locking in suprachiasmatic nucleus (SCN) circadian pacemakers, and AII amacrine-mediated rod pathway signaling in the retina; its knockout disrupts spike timing precision, impairs visuomotor reflexes, and increases seizure susceptibility. Mutations such as R278H destabilize gap junction plaques, leading to juvenile myoclonic epilepsy and Oculodentodigital dysplasia phenotypes.

References
[1] Srinivas M, et al. J Neurosci. 1999 Nov 15;19(22):9848-55. [2] Ding X, et al. Cell Discov. 2024 Jun 18;10(1):68.

PVDFメンブレン孔径参考表

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%