Anti-Phospho-E2F1 (Ser337) Polyclonal Antibody is a Rabbit antibody targeting Phospho-E2F1 (Ser337). Anti-Phospho-E2F1 (Ser337) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Mouse (predicted:Horse)

1. 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 (TMAB-10612) 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.
2. 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 (TMAB-10612), 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 1 X PBS containing 0.5% BSA.
Protocol
The cells werE. Primary antibody (TMAB-10612, 1 μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X 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 20,000 events was performed.
3. Blank control: Mosue spleen.
Primary Antibody (green line): Rabbit Anti-phospho-E2F1 (Ser337) antibody (TMAB-10612)
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℃. 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 20,000 events was performed.
4. Sample:
Lane 1: Skin (Mouse) Lysate at 30 μg
Lane 2: Spleen (Mouse) Lysate at 40 μg
Lane 3: Large intestine (Mouse) Lysate at 40 μg
Primary:
Anti-phospho-E2F1 (Ser337) (TMAB-10612) at 1/1000 dilution
Anti-beta-Actin 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
5. 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 (TMAB-10612) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
6. Sample:
Lane 1: Mouse Spleen tissue lysates
Lane 2: Mouse Colon tissue lysates
Primary: Anti-phospho-E2F1 (Ser337) (TMAB-10612) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46 kDa
Observed band size: 53 kDa

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100-500; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human E2F1 around the phosphorylation site of Ser337

Human

E2F1

Transcription factor E2F1

Transcription activator that binds DNA cooperatively with dp proteins through the E2 recognition site, 5'-TTTC[CG]CGC-3' found in the promoter region of a number of genes whose products are involved in cell cycle regulation or in DNA replication. The DRTF1/E2F complex functions in the control of cell-cycle progression from G1 to S phase. E2F-1 binds preferentially RB1 protein, in a cell-cycle dependent manner. It can mediate both cell proliferation and p53-dependent apoptosis.