p21 Recombinant Rabbit Monoclonal Antibody [PSH22-37]

☑ Relative expression (RE),Cell treatment (CT)

Western blot analysis of p21 on different lysates with Rabbit anti-p21 antibody (HA724295) at 1/2,000 dilution.

Lane 1: HCT 116 (Human colon cancer cells)
Lane 2: NCI-H1299 (Human non-small cell Lung carcinoma cells) (Negative)
Lane 3: A549 (Human lung adenocarcinoma cells)
Lane 4: MCF7 (Human breast cancer cells)
Lane 5: MCF7 (Human breast cancer cells) treated with Nutlin 3a (10uM, 24h)

Lysates/proteins at 10 µg/Lane.
Exposure time: 1 minutes; ECL: K1801

H1299 is a negative control (PMID: 23268708).

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA724295, 1/2,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 21 kDa
Observed band size: 21 kDa

☑ Cell treatment (CT)

Application: Immunocytochemistry (IF-cell)

Species: Human
Sample: MCF7 (Human cervix adenocarcinoma epithelial cell) treated or untreated with Nutlin 3a (10 μM, 24h)

Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
Permeabilization: 0.1% Triton X-100, 15 minutes at room temperature.

Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.
Primary antibody: HA724295, 1/5,000, overnight at 4℃.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.

Counterstain: Beta tubulin (HA601187, red), 1/100, overnight at 4℃. The Nuclear counterstain was DAPI (Blue).

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Colon cancer
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× PBST
Endogenous peroxidase blocking: 3% H2O2, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724295, 1/200, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Ovarian Carcinoma
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× PBST
Endogenous peroxidase blocking: 3% H2O2, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724295, 1/200, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Invasive Ductal Carcinoma of the Breast
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

Wash buffer: 1× PBST
Endogenous peroxidase blocking: 3% H2O2, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA724295, 1/200, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunoprecipitation (IP)

p21 was immunoprecipitated in 0.2 mg HCT 116 (Human colon cancer cell) cell lysate with HA724295 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA724295 at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HCT 116 cell lysate (input)
Lane 2: Rabbit IgG instead of HA724295 in HCT 116 cell lysate
Lane 3: HA724295 IP in HCT 116 cell lysate

Exposure time: 1 minute
Blocking: 5% NFDM/TBST, 1 hour at room temperature

Primary dilution: HA724295, 1/2,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature

Predicted band size: 21 kDa
Observed band size: 21 kDa