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Description biologique
| Spécificité | Ferritin Light Chain Antibody [N23F20] détecte les niveaux endogènes de la protéine totale Ferritin Light Chain. |
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| Contexte | La chaîne légère de ferritine (FTL), codée par le gène FTL sur le chromosome 19q13.33, est l'une des deux sous-unités (avec la chaîne lourde de ferritine, FTH) qui s'auto-assemblent en une nanocage sphérique creuse de 24 mérides (~450 kDa) servant de protéine de stockage de fer intracellulaire primaire chez les eucaryotes. Chaque monomère de FTL adopte un repliement globulaire compact avec un faisceau de cinq hélices α (hélices A-E) formant le cœur de la sous-unité ; 24 sous-unités FTL/FTH s'oligomérisent via des contacts inter-sous-unités hydrophobes aux axes de symétrie 4 fois (canaux d'entrée du fer ferrique) et 3 fois (pores de nucléation/libération du fer), créant une cavité centrale d'environ 80 Å de diamètre tapissée de résidus acides (Glu, Asp) qui nucléent la formation du noyau minéral de ferrihydrite. Contrairement au centre ferroxidase de FTH, la FTL manque de résidus catalytiques (Glu27, Tyr34, Glu62, Gln141 et Glu107 sont absents) mais facilite la précipitation du Fe(III) par l'intermédiaire de sites de liaison au phosphate de surface et de groupes de canaux de fer. La FTL favorise le stockage à long terme du fer en accélérant la nucléation de la ferrihydrite, en détoxifiant le pool de fer labile pour prévenir les dommages ROS médiés par Fenton, elle est régulée translationnellement par la liaison de l'élément sensible au fer (IRE)/IRP1 dans son UTR 5' ; les rapports H/L spécifiques aux tissus modulent la cinétique du fer (libération plus lente riche en L). Les mutations de FTL provoquent le syndrome d'hyperferritinémie-cataracte héréditaire (apoferritine régulée à la hausse), la neurodégénérescence avec accumulation de fer cérébral (NBIA3) et contribuent au cancer. |
Informations dutilisation
| Application | IHC, IF, FCM | Dilution |
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| Réactivité | Human | ||||||||
| Source | Rabbit Monoclonal Antibody | MW | |||||||
| Tampon de stockage | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Stockage (À partir de la date de réception) |
-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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Références
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Données dapplication
IF
Validé par Selleck
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Immunofluorescent analysis of Hela cells using F2743 (green, 1:50), Hoechst (blue) and tubulin (Red).