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Descripción biológica
| Especificidad | Ferritin Light Chain Antibody [N23F20] detecta niveles endógenos de la proteína total de Ferritin Light Chain. |
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| Antecedentes | La cadena ligera de ferritina (FTL), codificada por el gen FTL en el cromosoma 19q13.33, es una de las dos subunidades (junto con la cadena pesada de ferritina, FTH) que se autoensamblan en una nanocápsula esférica hueca de 24 meros (~450 kDa) que sirve como la principal proteína intracelular de almacenamiento de hierro en los eucariotas. Cada monómero de FTL adopta un plegamiento globular compacto con un haz de cinco hélices α (hélices A-E) que forman el núcleo de la subunidad; 24 subunidades de FTL/FTH oligomerizan a través de contactos hidrofóbicos inter-subunidad en los ejes de simetría 4-plegados (canales de entrada de hierro férrico) y 3-plegados (poros de nucleación/liberación de hierro), creando una cavidad central de ~80 Å de diámetro revestida por residuos ácidos (Glu, Asp) que nuclean la formación del núcleo mineral de ferrihidrita. A diferencia del centro ferroxidasa de FTH, FTL carece de residuos catalíticos (Glu27, Tyr34, Glu62, Gln141 y Glu107 están ausentes) pero facilita la precipitación de Fe(III) a través de sitios de unión a fosfato de superficie y grupos de canales de hierro. FTL promueve el almacenamiento de hierro a largo plazo acelerando la nucleación de ferrihidrita, desintoxicando el pool de hierro lábil para prevenir el daño por ROS mediado por Fenton, regula traslacionalmente mediante la unión del elemento de respuesta a hierro (IRE)/IRP1 en su 5' UTR; las relaciones H/L específicas de tejido modulan la cinética del hierro (liberación más lenta rica en L). Las mutaciones de FTL causan el síndrome hereditario de hiperferritinemia-catarata (apoferritina regulada al alza), neurodegeneración con acumulación de hierro cerebral (NBIA3) y contribuyen al cáncer. |
Información de uso
| Aplicación | IHC, IF, FCM | Dilución |
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| Reactividad | Human | ||||||||
| Fuente | Rabbit 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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Datos de aplicación
IF
Validado por Selleck
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Immunofluorescent analysis of Hela cells using F2743 (green, 1:50), Hoechst (blue) and tubulin (Red).