Phospho-E2F1 (Ser364) Rabbit Polyclonal Antibody

• Catalog Number: orb5095
• Category: Antibodies
• Description: Phospho-E2F1 (Ser364) Rabbit Polyclonal Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: FC, ICC, IF, IHC-Fr, IHC-P, WB
• Predicted Reactivity: Mouse
• Reactivity: Human, Rat
• Isotype: IgG
• Immunogen: KLH conjugated Synthesised phosphopeptide derived from human E2F1 around the phosphorylation site of Ser364 MG(p-S)LR
• Antibody Type: Primary Antibody
• Concentration: 1mg/ml
• Dilution range: WB=1:500-2000, IHC-P=1:100-500, IHC-F=1:100-500, ICC/IF=1:100, IF=1:100-500, Flow-Cyt=1ug/Test
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: 48 kDa
• Target: E2F1
• UniProt ID: Q01094
• RRID: AB_10947415
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Buffer/Preservatives: 0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
• Alternative names: E2F1(phospho S364); E2F1(phospho Ser364); p-E2F1(S
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Blank control (Black line): Molt4 (Black). Primary Antibody (green line): Rabbit Anti-phospho-E2F1 (Ser364) antibody (orb5095), dilution: 1 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647, dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.

Blank control: HepG2. Primary Antibody (green line): Rabbit Anti-phospho-E2F1 (Ser364) antibody (orb5095), dilution: 1 ug/Test, Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 ug/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.

HepG2 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37°C for 20 min, Antibody incubation with (phospho-E2F1 (Ser364)) polyclonal Antibody, Unconjugated (orb5095) 1:100, 90 minutes at 37°C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded (Human colon carcinoma), Antigen retrieval by microwave in sodium citrate buffer (pH6.0), Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes, Blocking buffer (3% BSA) at RT for 30 min, Antibody incubation with (phospho-E2F1 (Ser364)) Polyclonal Antibody, Unconjugated (orb5095) at 1:400 overnight at 4°C, followed by conjugation to the secondary antibody (labeled with HRP) and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat colon), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (phospho-E2F1 (Ser364)) Polyclonal Antibody, Unconjugated (orb5095) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat testis), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (phospho-E2F1 (Ser364)) Polyclonal Antibody, Unconjugated (orb5095) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Lane 1: Hela (Human) Cell Lysate at 30 ug, Lane 2: A673 (Human) Cell Lysate at 30 ug, Lane 3: Molt-4 (Human) Cell Lysate at 30 ug, Primary: Anti-phospho-E2F1 (Ser364) (orb5095) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 55-60 kD, Observed band size: 60 kD.