Anti-p53 抗体 [PAb 1801]

• ELISA

Unknown

ELISA - Anti-p53 antibody [PAb 1801] (AB28)

Analysis of recombinant p53 protein (ab43615) in MCF7 cells treated with vehicle (VEH)as well as MCF7 cells treated with camptothecin (CAM) and no material (NO) by sandwich ELISA with the use of anti-53 antibody (ab28) as capture and anti-p53 antibody (ab32389) as detector.

• WB

Lab

Western blot - Anti-p53 antibody [PAb 1801] (AB28)

All Lanes : Anti-p53 antibody [PAb 1801] (ab28) at 5 μg/ml

Lane 1 and 10 : Wild-type HCT116 cell lysate

Lane 2 and 11 : Wild-type HCT116 + irinotecan (10 μM, 24 hours) cell lysate

Lane 3 and 12 : p53 knockout HCT116 cell lysate

Lane 4 and 13 : p53 knockout HCT116 + irinotecan (10 μM, 24 hours) cell lysate

Lane 5 and 14 : A431 cell lysate (positive control)

Lane 6 and 15 : Saos-2 cell lysate (negative control)

Lane 7 and 16 : MEF cell lysate

Lane 8 and 17 : Wild-type HAP1 cell lysate

Lane 9 and 18 : p53 knockout HAP1 cell lysate

Lanes 1-9 : 1% BSA blocking buffer

Lanes 10-18 : 3% Milk blocking buffer

Secondary
Goat Anti-Mouse IgG H&L (HRP) at 1/5000 dilution

We recommend using 3% milk as the blocking agent for western blot.

All lanes:

Western blot - Anti-p53 antibody [PAb 1801] (ab28)

Predicted band size: 43 kDa

true

• WB

Unknown

Western blot - Anti-p53 antibody [PAb 1801] (AB28)

The image shows a Western blot for ab28, testing the threshold detection of the p53 antibody. The lanes are as follows :

Lane 1 - 20ug of A549 lysate, lane 2 - 10ug of A549 lysate, lane 3 - 5ug of A549 lysate, lane 4 - 2.5ug of A549 lysate, lane 5 - 1.5ug of A549 lysate.

This picture was submitted as part of the review by Craig Carson.

All lanes:

Western blot - Anti-p53 antibody [PAb 1801] (ab28)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-p53 antibody [PAb 1801] (AB28)

Lanes 1-9 : Merged (red and green) signal.

ab28 was shown to specifically react with p53 in wild-type HAP1 and HCT116 cells treated with irinotecan. No band was observed in p53 knockout HAP1 or HCT116 cells. Wild-type and p53 knockout samples, positive and negative controls were subjected to SDS-PAGE. ab28 and ab181602 (loading control to GAPDH) were diluted 5 μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-p53 antibody [PAb 1801] (ab28) at 5 µg/mL

Lane 1:

Wild-type HCT116 cell lysate at 30 µg

Lane 2:

Wild-type HCT116 + irinotecan (10 μM, 24 hours) cell lysate at 30 µg

Lane 3:

p53 knockout HCT116 cell lysate at 30 µg

Lane 4:

p53 knockout HCT116 + irinotecan (10 μM, 24 hours) cell lysate at 30 µg

Lane 5:

A431 cell lysate (positive control) at 20 µg

Lane 6:

Saos-2 cell lysate (negative control) at 20 µg

Lane 7:

MEF cell lysate at 20 µg

Lane 8:

Wild-type HAP1 cell lysate at 20 µg

Lane 9:

p53 knockout HAP1 cell lysate at 20 µg

Predicted band size: 43 kDa

false

• WB

Ap

Western blot - Anti-p53 antibody [PAb 1801] (AB28)

This blot was produced using a 10% 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 3% Milk before being incubated with ab28 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

We recommend using 3% milk as the blocking agent for Western blot.

All lanes:

Western blot - Anti-p53 antibody [PAb 1801] (ab28) at 5 µg/mL

All lanes:

MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/10000 dilution

Predicted band size: 43 kDa

Observed band size: 53 kDa,60 kDa

true

Exposure time: 20min