Anti-p53 antibody [DO-1] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

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 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) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (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™).

This data was developed using the same antibody clone in a different buffer formulation (ab1101).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 (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 (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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 (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 (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 Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 (ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

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.

This image was produced using the same antibody clone but in a different formulation; PBS and sodium azide (ab1101).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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 ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• ChIP

Lab

ChIP - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

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

This image was produced using the same antibody clone but in a different formulation; PBS and sodium azide (ab1101).

• WB

Lab

Western blot - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

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] (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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution. This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab1101).

All lanes:

Western blot - Anti-p53 antibody [DO-1] (<a href='/en-us/products/primary-antibodies/p53-antibody-do-1-chip-grade-ab1101'>ab1101</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/20000 dilution

Lanes 1 - 6:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (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] (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 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 12:

Western blot - Anti-p53 antibody [DO-1] (<a href='/en-us/products/primary-antibodies/p53-antibody-do-1-chip-grade-ab1101'>ab1101</a>) at 1/1000 dilution

Lanes 1 - 12:

Western blot - 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 (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/20000 dilution

false

• WB

Lab

Western blot - Anti-p53 antibody [DO-1] - BSA and Azide free (AB237976)

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] (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 cell lysates with no signal observed at this size in tp53 knockout cell line ab276092 (knockout cell lysate ab282999). 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-p53 antibody [DO-1] (<a href='/en-us/products/primary-antibodies/p53-antibody-do-1-chip-grade-ab1101'>ab1101</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human TP53 (p53) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-tp53-p53-knockout-a549-cell-line-ab276092'>ab276092</a>) at 20 µg

Lane 3:

Wild-type Jurkat cell lysate at 20 µg

Lane 4:

Western blot - Human TP53 (p53) knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-tp53-p53-knockout-jurkat-cell-line-ab276112'>ab276112</a>) at 20 µg

Lane 5:

A431 cell lysate at 20 µg

Lane 6:

Saos-2 cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 49 kDa

false