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Descripción biológica
| Especificidad | Poly/Mono-ADP Ribose Antibody [A22J18] reconoce los niveles endógenos de proteínas ADP ribosiladas y no presenta reactividad cruzada con otras modificaciones post-traduccionales. |
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| Antecedentes | El poli(ADP-ribosa) (PAR) y la mono(ADP-ribosa) (MAR) son modificaciones post-traduccionales reversibles mediadas por ADP-ribosiltransferasas, particularmente miembros de la familia de la poli(ADP-ribosa) polimerasa (PARP). MAR se refiere a la adición covalente de una única unidad de ADP-ribosa —transferida de NAD⁺— a residuos de aminoácidos específicos (comúnmente aspartato, glutamato o lisina) en proteínas diana. En contraste, PAR consiste en dos o más unidades de ADP-ribosa unidas a través de enlaces glicosídicos ribosa-ribosa, formando cadenas lineales o ramificadas. Estructuralmente, cada unidad de ADP-ribosa contribuye con ~0.5 kDa, lo que convierte a PAR en un polímero grande, altamente cargado negativamente y flexible, capaz de servir de andamiaje para diversos ensamblajes proteicos. PARP1, la PARP nuclear más abundante y catalíticamente activa, se activa rápidamente por rupturas de cadenas de ADN y señales de estrés, catalizando la PARilación para reclutar y organizar complejos de DNA Damage/DNA Repair. La MARilación y la PARilación desempeñan roles celulares distintos: MAR a menudo regula la actividad o las interacciones de proteínas individuales, mientras que PAR cumple funciones más amplias como elemento estructural dinámico en procesos como el DNA Damage/DNA Repair, la remodelación de la cromatina, la formación de gránulos de estrés, el ensamblaje del huso mitótico y la integridad nucleolar. El equilibrio entre la síntesis por PARPs y la degradación por enzimas como la poli(ADP-ribosa) glicohidrolasa (PARG) y las hidrolasas de macrodominios es fundamental para mantener la homeostasis celular. |
Información de uso
| Aplicación | WB, IF | Dilución |
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| Reactividad | All | ||||||
| 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 | ||||
| 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 59°C protected from light.
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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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