Poly/Mono-ADP Ribose Antibody [A22J18]

Toepassing: Reactiviteit:All

Klantenbeoordeling

Gebruiksinformatie

Verdunning
WB1:1000 IF1:12000 - 1:48000

Toepassing
WB, IF

Reactiviteit
All

Bron
Rabbit Monoclonal Antibody

Opslagbuffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Opslag (vanaf ontvangstdatum)
-20°C (avoid freeze-thaw cycles), 2 years

Experimentele methoden

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.

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.

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：

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.

Datasheet & SDS

Biologische beschrijving

Specificiteit
Poly/Mono-ADP Ribose Antibody [A22J18] recognizes endogenous levels of ADP ribosylated proteins and does not cross-react with other post translational modifications.

Kloon
A22J18

Synoniem
Poly (ADP-Ribose) Polymer,Poly/Mono-ADP Ribose

Achtergrond
Poly(ADP-ribose) (PAR) and mono(ADP-ribose) (MAR) are reversible post-translational modifications mediated by ADP-ribosyltransferases, particularly members of the poly(ADP-ribose) polymerase (PARP) family. MAR refers to the covalent addition of a single ADP-ribose unit—transferred from NAD⁺—to specific amino acid residues (commonly aspartate, glutamate, or lysine) on target proteins. In contrast, PAR consists of two or more ADP-ribose units linked through ribose–ribose glycosidic bonds, forming linear or branched chains. Structurally, each ADP-ribose unit contributes ~0.5 kDa, making PAR a large, highly negatively charged and flexible polymer capable of scaffolding diverse protein assemblies. PARP1, the most abundant and catalytically active nuclear PARP, is rapidly activated by DNA strand breaks and stress signals, catalyzing PARylation to recruit and organize DNA repair complexes. MARylation and PARylation play distinct cellular roles—MAR often regulates individual protein activity or interactions, while PAR serves broader functions as a dynamic structural element in processes such as DNA repair, chromatin remodeling, stress granule formation, mitotic spindle assembly, and nucleolar integrity. The balance between synthesis by PARPs and degradation by enzymes like poly(ADP-ribose) glycohydrolase (PARG) and macrodomain hydrolases is critical for maintaining cellular homeostasis.

Achtergrond

Poly(ADP-ribose) (PAR) and mono(ADP-ribose) (MAR) are reversible post-translational modifications mediated by ADP-ribosyltransferases, particularly members of the poly(ADP-ribose) polymerase (PARP) family. MAR refers to the covalent addition of a single ADP-ribose unit—transferred from NAD⁺—to specific amino acid residues (commonly aspartate, glutamate, or lysine) on target proteins. In contrast, PAR consists of two or more ADP-ribose units linked through ribose–ribose glycosidic bonds, forming linear or branched chains. Structurally, each ADP-ribose unit contributes ~0.5 kDa, making PAR a large, highly negatively charged and flexible polymer capable of scaffolding diverse protein assemblies. PARP1, the most abundant and catalytically active nuclear PARP, is rapidly activated by DNA strand breaks and stress signals, catalyzing PARylation to recruit and organize DNA repair complexes. MARylation and PARylation play distinct cellular roles—MAR often regulates individual protein activity or interactions, while PAR serves broader functions as a dynamic structural element in processes such as DNA repair, chromatin remodeling, stress granule formation, mitotic spindle assembly, and nucleolar integrity. The balance between synthesis by PARPs and degradation by enzymes like poly(ADP-ribose) glycohydrolase (PARG) and macrodomain hydrolases is critical for maintaining cellular homeostasis.

Referenties
https://pubmed.ncbi.nlm.nih.gov/24914234/

Eiwitgrootte	PVDF-transfermembraan Poriemaat
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Eiwitgrootte	Scheidingsgelpercentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%