Poly/Mono-ADP Ribose Antibody [A22J18]

Katalog-Nr. F0957

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Biologische Beschreibung

Spezifität

Poly/Mono-ADP Ribose Antibody [A22J18] erkennt endogene Spiegel ADP-ribosylierter Proteine und kreuzreagiert nicht mit anderen posttranslationalen Modifikationen.
Hintergrund Poly(ADP-Ribose) (PAR) und Mono(ADP-Ribose) (MAR) sind reversible posttranslationale Modifikationen, die durch ADP-Ribosyltransferasen, insbesondere Mitglieder der Poly(ADP-Ribose)-Polymerase (PARP)-Familie, vermittelt werden. MAR bezeichnet die kovalente Addition einer einzelnen ADP-Ribose-Einheit – übertragen von NAD⁺ – an spezifische Aminosäurereste (häufig Aspartat, Glutamat oder Lysin) auf Zielproteinen. Im Gegensatz dazu besteht PAR aus zwei oder mehr ADP-Ribose-Einheiten, die durch Ribose-Ribose-Glykosidbindungen miteinander verbunden sind und lineare oder verzweigte Ketten bilden. Strukturell trägt jede ADP-Ribose-Einheit ~0,5 kDa bei, was PAR zu einem großen, stark negativ geladenen und flexiblen Polymer macht, das in der Lage ist, verschiedene Proteinkomplexe zu gerüsten. PARP1, die am häufigsten vorkommende und katalytisch aktivste nukleäre PARP, wird schnell durch DNA-Strangbrüche und Stresssignale aktiviert und katalysiert die PARylierung, um DNA-Reparaturkomplexe zu rekrutieren und zu organisieren. MARylierung und PARylierung spielen unterschiedliche zelluläre Rollen – MAR reguliert oft die Aktivität oder Interaktionen einzelner Proteine, während PAR umfassendere Funktionen als dynamisches Strukturelement in Prozessen wie DNA Damage/DNA Repair, Chromatin-Remodeling, Stressgranula-Bildung, mitotischer Spindelmontage und nukleolärer Integrität erfüllt. Das Gleichgewicht zwischen der Synthese durch PARPs und dem Abbau durch Enzyme wie Poly(ADP-Ribose)-Glykohyalase (PARG) und Makrodomänenhydrolasen ist entscheidend für die Aufrechterhaltung der zellulären Homöostase.

Nutzungsinformationen

Anwendung WB, IF Verdünnung
WB IF
1:1000 1:12000 - 1:48000
Reaktivität All
Quelle Rabbit 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
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.
 
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/24914234/

Anwendungsdaten