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Descripción biológica
| Especificidad | Connexin 36 Antibody [L23J6] detecta niveles endógenos de proteína total Connexin 36. |
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| Antecedentes | La conexina 36 (Cx36, GJC1) es una proteína de unión gap específica de neuronas altamente expresada en interneuronas del cerebro de mamíferos y neuronas retinianas, que se ensambla en conexones hexaméricos donde cada monómero presenta cuatro dominios transmembrana (TM1–4); TM1 y TM2 forman la constricción hidrofóbica que recubre el poro, flanqueada por bucles extracelulares (EL1/EL2) que contienen d-prolinas conservadas (Pro47, Pro187) que facilitan el acoplamiento intercelular. El canal posee una cola N-terminal intracelular corta con sitios de fosforilación de CaMKII (notablemente Ser293), y un dominio de bucle citoplasmático (CL) que media la unión de Ca²⁺/calmodulina, regulando alostéricamente el poro acuoso de ~15 Å de diámetro, que es selectivamente permeable a cationes y aniones de menos de 1,2 kDa (incluyendo K⁺, IP₃, cAMP y amarillo de Lucifer). Aunque insensibles al voltaje, los canales de unión gap Cx36 son sensibles al pH y al Ca²⁺, exhiben una baja conductancia unitaria (~15 pS) y median un acoplamiento electrotónico bidireccional para una sincronía precisa de los spikelets a través de las redes inhibidoras al permitir el flujo directo de corriente entre somas y dendritas. La fosforilación dependiente de CaMKII en Ser293 aumenta la probabilidad de apertura durante la plasticidad sináptica, mientras que agentes farmacológicos como la mefloquina y la quinina inhiben la conductancia del canal al unirse a las bolsas hidrofóbicas inter-monoméricas TM1/TM2 (I35/V38/A39/I40, I76/V80), formando anillos oclusivos que deshidratan la vía de permeación iónica y reducen la conductancia en más del 50%. La Cx36 es esencial para las oscilaciones gamma, el enclavamiento de fase en los marcapasos circadianos del núcleo supraquiasmático (SCN) y la señalización de la vía de bastones mediada por las amacrinas AII en la retina; su eliminación interrumpe la precisión del tiempo de espigas, altera los reflejos visuomotores y aumenta la susceptibilidad a las convulsiones. Mutaciones como R278H desestabilizan las placas de unión gap, lo que lleva a fenotipos de epilepsia mioclónica juvenil y displasia oculodentodigital. |
Información de uso
| Aplicación | IHC, IF | Dilución |
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| Reactividad | Human, Mouse | ||||||
| Fuente | Mouse Monoclonal Antibody | MW | |||||
| Tampón de almacenamiento | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Almacenamiento (Desde la fecha de recepción) |
-20°C (avoid freeze-thaw cycles), 2 years | ||||
| 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.
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| 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.
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Referencias
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