Phospho-E2F1 (Ser337) Rabbit Polyclonal Antibody

• Catalog Number: orb5096
• Category: Antibodies
• Description: Phospho-E2F1 (Ser337) Rabbit Polyclonal Antibody
• Target: E2F1
• Clonality: Polyclonal
• Species/Host: Rabbit
• Isotype: IgG
• Conjugation: Unconjugated
• Reactivity: Human, Mouse
• Predicted Reactivity: Equine
• Form/Appearance: Liquid
• Concentration: 1mg/ml
• Buffer/Preservatives: 0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
• Purification: Affinity purified by Protein A
• Immunogen: KLH conjugated Synthesised phosphopeptide derived from human E2F1 around the phosphorylation site of Ser337 PS(p-S)PP
• UniProt ID: Q01094
• RRID: AB_10925990
• MW: 46 kDa
• Tested applications: FC, ICC, IF, IHC-Fr, IHC-P, WB
• Dilution range: WB=1:500-2000, IHC-P=1:100-500, IHC-F=1:100-500, ICC/IF=1:100-500, IF=1:100-500, Flow-Cyt=1ug/Test
• Antibody Type: Primary Antibody
• 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.
• Alternative names: phospho-E2F1(Ser337); p-E2F1(phospho-Ser337); E2F1
• Research Area: Cancer Research, Cell Biology, Forkhead box (FOX)
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Blank control (blue): TM4 cells (fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice). Primary Antibody: Rabbit Anti-E2F 1 antibody (orb5096), dilution: 1 µg in 100 µl 1X PBS containing 0.5% BSA, Isotype Control Antibody: Rabbit IgG (orange), used under the same conditions, Secondary Antibody: Goat anti-rabbit IgG-PE (white blue), dilution: 1:200 in 1X PBS containing 0.5% BSA. Protocol, The cells were Primary antibody (orb5096, 1 µg/1x10^6 cells) were incubated for 30 min on the ice, followed by 1X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20000 events was performed.

Blank control: Mosue spleen. Primary Antibody (green line): Rabbit Anti-phospho-E2F1 (Ser337) antibody (orb5096), dilution: 2 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-FITC, 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 -20°C. 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 (Ser337)) polyclonal Antibody, Unconjugated (orb5096) 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 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 (E2F1 (Ser337)) Polyclonal Antibody, Unconjugated (orb5096) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Lane 1: Mouse Spleen tissue lysates, Lane 2: Mouse Colon tissue lysates, Primary: Anti-phospho-E2F1 (Ser337) (orb5096) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 46 kDa, Observed band size: 53 kDa.

Sample: Lane 1: Skin (Mouse) Lysate at 30 ug, Lane 2: Spleen (Mouse) Lysate at 40 ug, Lane 3: Large intestine (Mouse) Lysate at 40 ug, Primary: Anti-phospho-E2F1 (Ser337) (orb5096) at 1/1000 dilution, Anti-beta-Actin (orb500817) at 1/2000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 55-60 kD, Observed band size: 58 kD.