Connexin 36 Antibody [L23J6]

Katalog-Nr. F3937

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Biologische Beschreibung

Spezifität Connexin 36 Antibody [L23J6] weist endogene Spiegel des gesamten Connexin 36-Proteins nach.
Hintergrund Connexin 36 (Cx36, GJC1) ist ein neuronspezifisches Gap-Junction-Protein, das in Interneuronen und retinalen Neuronen des Säugetiergehirns stark exprimiert wird und sich zu hexameren Connexonen zusammensetzt, wobei jedes Monomer vier Transmembrandomänen (TM1–4) aufweist; TM1 und TM2 bilden die porenkleidende hydrophobe Verengung, flankiert von extrazellulären Schleifen (EL1/EL2), die konservierte d-Proline (Pro47, Pro187) enthalten, die das interzelluläre Andocken erleichtern. Der Kanal besitzt einen kurzen intrazellulären N-terminalen Schwanz mit CaMKII-Phosphorylierungsstellen (insbesondere Ser293) und eine zytoplasmatische Schleifendomäne (CL), die die Ca²⁺/Calmodulin-Bindung vermittelt und die wässrige Pore mit einem Durchmesser von ~15 Å allosterisch steuert, die selektiv für Kationen und Anionen unter 1,2 kDa (einschließlich K⁺, IP₃, cAMP und Lucifergelb) permeabel ist. Während spannungsunempfindlich, sind Cx36-Gap-Junction-Kanäle pH- und Ca²⁺-empfindlich, weisen eine geringe unitäre Leitfähigkeit (~15 pS) auf und vermitteln eine bidirektionale elektrotonische Kopplung für präzise Spikelet-Synchronität in inhibitorischen Netzwerken, indem sie einen direkten Stromfluss zwischen Somata und Dendriten ermöglichen. Die CaMKII-abhängige Phosphorylierung an Ser293 erhöht die offene Wahrscheinlichkeit während der synaptischen Plastizität, während pharmakologische Wirkstoffe wie Mefloquin und Chinin die Kanalleitfähigkeit hemmen, indem sie hydrophobe Taschen (I35/V38/A39/I40, I76/V80) zwischen den Monomeren TM1/TM2 binden und okklusive Ringe bilden, die den Ionenpermeationsweg dehydrieren und die Leitfähigkeit um über 50 % reduzieren. Cx36 ist essentiell für Gamma-Oszillationen, Phasenverriegelung in suprachiasmatischen Kern (SCN)-zirkadianen Schrittmachern und AII-Amakrin-vermittelte Stäbchenwegsignalisierung in der Netzhaut; sein Knockout stört die Präzision des Spike-Timings, beeinträchtigt visuomotorische Reflexe und erhöht die Anfallsempfindlichkeit. Mutationen wie R278H destabilisieren Gap-Junction-Plaques und führen zu Phänotypen der juvenilen myoklonischen Epilepsie und der okulodentodigitalen Dysplasie.

Nutzungsinformationen

Anwendung IHC, IF Verdünnung
IHC IF
1:160-1:250 1:160-1:250
Reaktivität Human, Mouse
Quelle Mouse Monoclonal Antibody MW
Lagerpuffer PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3
Lagerung
(Ab dem Datum des Erhalts)		 -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.
 
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.
 

Referenzen

  • https://pubmed.ncbi.nlm.nih.gov/10559394/
  • https://pubmed.ncbi.nlm.nih.gov/38890333/

Anwendungsdaten