ab1101 staining p53 - ChIP Grade in A431 cells. 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 5µg/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).

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™).

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

All lanes : 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 why is the actual band size different from the predicted?

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.

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

All lanes :

Lanes 1 & 5 & 9 : Wild-type HAP1 cell lysate at 20 µg
Lanes 2 & 6 & 10 : p53 knockout HAP1 cell lysate at 20 µg
Lanes 3 & 7 : A431 cell lysate at 20 µg
Lanes 4 & 8 & 12 : Saos-2 cell lysate at 20 µg
Lane 11 : A431 cell lysate

Predicted band size: 43 kDa

Lanes 1, 2, 3 and 4: Green signal from target – ab1101 observed at 53 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control – ab181602 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab1101 was shown to specifically react with p53 in wild type HAP1 cells No band was observed in p53 knockout HAP1 cell lysates. Wild type and p53 knockout samples were subjected to SDS-PAGE. ab1101 and ab181602 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4ºC. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

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

Lane 1 : HEK-293 (human embryonic kidney cell line) whole cell lysate
Lane 2 : DU 145 (human prostate carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 43 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

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 ab175783 at a 1/10,000 dilution for 1 hour at room temperature and then imaged using the Licor Odyssey CLx.

All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 2.5 µg/ml

Lane 1 : MDA-MB-361 (human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : A549 (human lung adenocarcinoma epithelial cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 43 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa (possible post-translational modification)

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab1101 (red line). The cells were fixed with 4% paraformaldehyde for 10 minutes and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab1101, 1/50 dilution) for 30 minutes at 22ºC. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) ab96879 at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] ab91361, 1 µg/1 x 10⁶ cells, used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.