Anti-p53 antibody [DO-1] - ChIP Grade

16 product images

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  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab1101 overnight at 4°C. Antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772 at a 1/10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.
  

  Image shows merged signal (red and green). Green - ab1101 observed at 53 kDa. Red - GAPDH loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602, observed at 37 kDa using as secondary Goat Anti-Rabbit IgG H&L (IRDye^® 680RD)- Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777

  All lanes: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution

  Lane 1: Wild-type HAP1 cell lysate (20 µg)

  Lane 2: p53 knockout HAP1 cell lysate (20 µg)

  Lane 3: A431 cell lysate (20 µg)

  Lane 4: Saos-2 cell lysate (20 µg)

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/10000 dilution

  Predicted band size: 36 kDa

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  ChIP - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Chromatin was prepared from HEK-293 (human epithelial cell line from embryonic kidney) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 2 μg of ab1101, and 10 ml of protein A sepharose beads,10 ml of protein G sepharose beads. No IgG was added to the beads control. The immunoprecipitated DNA was quantified by real time PCR. Primers were as follows:
  

  Bax-1, forward: GGGTTATCTCTTGGGCTCACAA.

  Bax-1, reverse: GAGCTCTCCCCAGCGCA.

  Bax-2, forward: TGG AGC TGC AGA GGA TGA TTG

  Bax-2, reverse: CCA GTT GAA GTT GCC GTC AGA

  PUMA, forward: ATG CCT GCC TCA CCT TCA TC

  PUMA, reverse: TCA CAC GTC GCT CTC TCT AAA CC

  p21-1, forward: GCT GTG GCT CTG ATT GGC TTT

  p21-1, reverse: ACA GGC AGC CCA AGG ACA AA

  p21-2, forward: CAT CCC CAC AGC AGA GGA GAA

  p21-2, reverse: ACC CAG GCT TGG AGC AGC TA

  p21-3, forward: GAG TCC TGT TTG CTT CTG GGC A

  p21-3, reverse: CTG CAT TGG GGC TGC CTA TGT A

  PCNA, forward: CCA CCA TAA AGC TGG GGC TT

  PCNA, reverse: TCT CCC CGC CTC TTT GAC TC

• 

  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 49 kDa in wild-type A549 and Jurkat cell lysates with no signal observed at this size in tp53 knockout cell lines Human TP53 (p53) knockout A549 cell line ab276092, Human TP53 (p53) knockout Jurkat cell line ab276112 (knockout cell lysates ab282999, Human TP53 (p53) knockout Jurkat cell lysate ab283832). To generate this image, wild-type and tp53 knockout A549 and Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.

  All lanes: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lanes 1 - 6: Western blot - Human TP53 (p53) knockout A549 cell line (Human TP53 (p53) knockout A549 cell line ab276092)

  Lane 2: TP53 knockout A549 cell lysate at 20 µg

  Lane 2: Western blot - Human TP53 (p53) knockout Jurkat cell line (Human TP53 (p53) knockout Jurkat cell line ab276112)

  Lane 3: Wild-type Jurkat cell lysate at 20 µg

  Lane 4: TP53 knockout Jurkat cell lysate at 20 µg

  Lane 5: A431 cell lysate at 20 µg

  Lane 6: Saos-2 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 43 kDa

  Observed band size: 49 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  ab1101 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab1101 at 1μg/ml concentration and Anti-beta Tubulin antibody - Loading Control ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
  

  Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

• 

  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Developed using the ECL technique with 30 second exposure.
  Blocking: 5% milk in PBS for 3 hours at RT.
  Primary antibody in 5% BSA + PBS was incubated overnight at 4°C.
  Secondary antibody in 5% milk + PBS was incubated for 2 hours at RT.

  All lanes: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 0.4 µg/mL

  Lane 1: Extract of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells incubated with vehicle at 20 µg

  Lane 2: Extract of HEK-293T cells incubated with etoposide at 20 µg

  Lane 3: Extract of HEK-293T cells incubated with etoposide at 7.5 µg

  Lane 4: Lambda phosphatase (diluted 1/400)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg

  Lane 5: Lambda phosphatase (diluted 1/100)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg

  Lane 6: Lambda phosphatase (diluted 1/25)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg

  Lane 7: Extract of MCF7 (human breast adenocarcinoma cell line) cells incubated with vehicle at 20 µg

  Lane 8: Extract of MCF7 cells incubated 6 hours with camptothecin at 20 µg

  Lane 9: Extract of MCF7 cells incubated 16 hours with camptothecin at 20 µg

  Lane 10: Extract of MCF7 cells incubated 24 hours with camptothecin at 20 µg

  Secondary

  All lanes: Goat anti mouse IgG(H&L)-HRP at 1/10000 dilution

  Predicted band size: 43 kDa

  Observed band size: 53 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  IHC image of ab1101 staining p53 in human colon adenocarcinoma formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab1101, 1/500 dilution, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. High magnification of the tumor region - T (lower right panel) and adjacent normal crypts - N (lower left panel) are shown.
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab1101 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) ab175783 at a 1/10,000 dilution for 1 hour at room temperature and then imaged using the Licor Odyssey CLx.

  All lanes: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution

  Lane 1: HEK-293 (human embryonic kidney cell line) whole cell lysate at 20 µg

  Lane 2: DU 145 (human prostate carcinoma cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution

  Predicted band size: 43 kDa

  Observed band size: 50 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  ab1101 staining mutant p53 in A431 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 100% methanol (5 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• 

  Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Flow cytometry overlay histogram showing wild-type p53 in Hek-293 positive cells (left) and MCF7 negative cells (right) stained with ab1101 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.2μg/ml (1/5000)) for 30min at 22°C. The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (Mouse IgG2a, Kappa Monoclonal [MOPC-173] -Isotype Control - ChIP Grade ab18413) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in Hek-293 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• 

  Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Flow cytometry overlay histogram showing mutant p53 in A-431 cells stained with ab1101 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.04μg/ml (1/25000)) for 30min at 22°C. The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (Mouse IgG2a, Kappa Monoclonal [MOPC-173] -Isotype Control - ChIP Grade ab18413) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in A-431 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

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  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Negative control: HL-60 (p53 null), NCI-H1299 (p53 null).

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 21941372, PMID: 21779513, PMID: 25996291).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) staining at 1/1000 dilution.

  In Western blot, Anti-p53 antibody [EPR17343] (Anti-p53 antibody [EPR17343] ab179477) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] (Anti-TP53 (phospho S376+S377+S392) antibody [RM1161] ab322465) at 1/1000 dilution

  Lane 1: Untreated HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: HCT 116 treated with 20uM etoposide for 15 hours whole cell lysate at 20 µg

  Lane 3: Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: A549 treated with 500nM doxorubicin for 24 hours whole cell lysate at 20 µg

  Lane 5: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 6: HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

  Lane 7: NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 53 kDa, 26-48 kDa, 36 kDa

  Exposure time: 10s

• 

  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 40/50 kDa in treated wild-type A549 cell lysates with no signal observed at this size in tp53 knockout cell line Human TP53 (p53) knockout A549 cell line ab276092. To generate this image, wild-type and tp53 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.

  Lanes 1 - 12: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution

  Lanes 1 - 12: Western blot - Anti-p53 antibody [DO-1] - BSA and Azide free (Anti-p53 antibody [DO-1] - BSA and Azide free ab237976)

  Lane 1: MCF7 Treated Camptothecin (20 ug/ml, 0 h) cell lysate at 15 µg

  Lane 2: MCF7 Treated Camptothecin (20 ug/ml, 6 h) cell lysate at 15 µg

  Lane 3: MCF7 Treated Camptothecin (20 ug/ml, 24 h) cell lysate at 15 µg

  Lane 4: Wild-type A549 Treated Camptothecin (20 ug/ml, 0 h) cell lysate at 15 µg

  Lane 5: Wild-type A549 Treated Camptothecin (20 ug/ml, 6 h) cell lysate at 15 µg

  Lane 6: Wild-type A549 Treated Camptothecin (20 ug/ml, 24 h) cell lysate at 15 µg

  Lane 7: TP53 knockout A549 Treated Camptothecin (20 ug/ml, 0 h) cell lysate at 15 µg

  Lane 8: TP53 knockout A549 Treated Camptothecin (20 ug/ml, 6 h) cell lysate at 15 µg

  Lane 9: TP53 knockout A549 Treated Camptothecin (20 ug/ml, 24 h) cell lysate at 15 µg

  Lane 10: Saos-2 cell lysate at 15 µg

  Lane 11: SK-BR-3 cell lysate at 15 µg

  Lane 12: A431 cell lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Performed under reducing conditions.

• 

  Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 50 kDa in wild-type HAP1 cell lysate with no signal observed at this size in tp53 knockout cell line. To generate this image, wild-type and tp53 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.

  All lanes: Western blot - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution

  Lane 1: Saos-2 cell lysate at 20 µg

  Lane 2: A431 cell lysate

  Lane 3: Wild-type HAP1 cell lysate at 20 µg

  Lane 4: TP53 knockout HAP1 cell lysate at 20 µg

  Lane 5: MCF7 cell lysate at 20 µg

  Lane 6: HEK-293T cell lysate at 20 µg

  Secondary

  Lanes 1 - 6: Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution

  Lanes 1 - 6: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 43 kDa

  Observed band size: 50 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  ab1101 staining wild-type p53 in MCF7 cells (a low expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  ab1101 staining wild-type p53 in Hek293 cells (a high expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
  
  Also suitable in cells fixed with 4% paraformaldehyde (10 min).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• 

  Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)

  Flow cytometry overlay histogram showing mutant p53 in wild-type HAP1 (green line) and TP53 knockout HAP1 cells (red line) stained with ab1101. The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.04 μg/ml (1/25000)) for 30min at 22°C.The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (Mouse IgG2a, Kappa Monoclonal [MOPC-173] -Isotype Control - ChIP Grade ab18413) was used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line, TP53 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in HAP1 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.