p53 Recombinant Mouse Monoclonal Antibody [6B5-R]

☑ Cell treatment (CT)

Western blot analysis of p53 on different lysates with Mouse anti-p53 antibody (HA601315) at 1/1,000 dilution.

Lane 1: 293T cell lysate
Lane 2: HT-29 cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 0.5μM doxorubicincell for 24 hours lysate
Lane 5: Neuro-2a cell lysate
Lane 6: COS-1 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: Lane 1-2: 1 minute 40 seconds; Lane 3-6: 3 minutes;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601315) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Relative expression (RE)

Western blot analysis of p53 on different lysates with Mouse anti-p53 antibody (HA601315) at 1/2,000 dilution.

Lane 1: A431 cell lysate
Lane 2: MDA-MB-231 cell lysate
Lane 3: Saos-2 cell lysate (negative)

Lysates/proteins at 30 µg/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: 6 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601315) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of p53 on different lysates with Mouse anti-p53 antibody (HA601315) at 1/2,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-p53 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: 40 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601315) at 1/2,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of A431 cells labeling p53 with Mouse anti-p53 antibody (HA601315) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-p53 antibody (HA601315) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.