Insulin Antibody [K13G15]

N.º de catálogo F0436

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Descripción biológica

Especificidad

Insulin Antibody [K13G15] reconoce los niveles endógenos de la proteína Insulin total.
Antecedentes

La insulina es una hormona peptídica compuesta por 51 aminoácidos, secretada por las células β del páncreas. Desempeña un papel vital en el mantenimiento de la homeostasis de la glucosa al unirse al receptor de insulina (IR), una Protein Tyrosine Kinase transmembrana compuesta por dos subunidades α extracelulares responsables de la unión del ligando y dos subunidades β intracelulares que median la transducción de señales. Tras la unión de la insulina, el receptor sufre autofosforilación en sus residuos de tirosina, activando su actividad quinasa. Esto conduce a la fosforilación de los sustratos del receptor de insulina (IRS), que a su vez reclutan y activan la fosfoinosítido 3-quinasa (PI3K). La PI3K activada genera fosfatidilinositol (3,4,5)-trifosfato (PIP3), que facilita la activación de AKT (protein kinase B). La AKT activada regula varios procesos metabólicos posteriores: promueve la captación de glucosa en el músculo esquelético y el tejido adiposo al inducir la translocación del transportador de glucosa GLUT4 a la membrana celular, suprime la producción hepática de glucosa al fosforilar e inhibir el factor de transcripción FOXO1, y mejora el almacenamiento de glucógeno al inhibir la glucógeno sintasa quinasa 3 (GSK3). Además, la señalización de la insulina inhibe la lipólisis a través de la supresión mediada por PDE3B y ABHD15 de la lipasa de triglicéridos del adipocito (ATGL) y la lipasa sensible a hormonas (HSL), mientras que simultáneamente promueve la lipogénesis y la síntesis de proteínas a través de la activación del complejo mTORC1. La resistencia a la insulina ocurre cuando las células se vuelven menos sensibles a la señalización de la insulina, a menudo debido a deficiencias en múltiples niveles de la vía. Estas pueden incluir una disminución en el número de receptores de insulina, una fosforilación defectuosa de IRS o una señalización PI3K/AKT alterada. Los factores contribuyentes incluyen la inflamación crónica, el estrés oxidativo y la acumulación de intermediarios lipídicos como los diacilgliceroles y las ceramidas, que interfieren con la actividad de IRS-1 y AKT. Además, la deposición ectópica de lípidos en tejidos como el hígado y el músculo esquelético puede activar quinasas relacionadas con el estrés, incluyendo JNK y PKCθ, lo que lleva a la fosforilación de serina de las proteínas IRS y la subsiguiente inhibición de la señalización de la insulina. La disfunción mitocondrial y el estrés del retículo endoplásmico (RE) también contribuyen al deterioro del metabolismo de la glucosa, impulsando la hiperglucemia persistente y la disfunción progresiva de las células β – características de la diabetes tipo 2.

Información de uso

Aplicación WB, IHC, IF, FCM Dilución
WB IHC IF FCM
1:200 1:6400 - 1:25600 1:200 - 1:800 1:50 - 1:200
Reactividad Human, Mouse, Rat
Fuente Rabbit Monoclonal Antibody MW 12 kDa
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
IF
Experimental Protocol：
 
Specimen Preparation 
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
NOTE: Paraformaldehyde is toxic, use only in a fume hood.
2. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.
 
Immunostaining
1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Mount slides usingmounting medium with DAPI and cover with coverslips.
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°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.
 
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.
 

Referencias

  • https://pubmed.ncbi.nlm.nih.gov/30067154/

Datos de aplicación

WB

Validado por Selleck

  • F0436-wb
    Lane 1: INS-1