Antibody Type: Rabbit Polyclonal

Application: WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

Reactivity: Human,Mouse,Rat (predicted:Dog,Pig,Cow,Horse,Rabbit,Sheep)

IgG

Human,Mouse,Rat (predicted:Dog,Pig,Cow,Horse,Rabbit,Sheep)

1. U2OS 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 (ATF4) polyclonal Antibody, Unconjugated (TMAB-00159) 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 nucleus.
2. Sample:
Cerebrum (Rat) Lysate at 40 μg
Cerebrum (Mouse) Lysate at 40 μg
Primary: Anti-ATF4 (TMAB-00159) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 37/50 kDa
Observed band size: 50 kDa
3. Blank control (blue line): Hela
Primary Antibody (green line): Rabbit Anti-Bid antibody (TMAB-00159)
Dilution: 0.2 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1 μg/test.
Protocol
The cells were fixed with 70% ethanol (Overnight at 4°C) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2% BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature.
4. Blank control: Mouse spleen.
Primary Antibody (green line): Rabbit Anti-ATF4 Alpha antibody (TMAB-00159)
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.
5. Hela 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 (ATF4) polyclonal Antibody, Unconjugated (TMAB-00159) 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 nucleus.
6. Paraformaldehyde-fixed, paraffin embedded (Human brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
7. Paraformaldehyde-fixed, paraffin embedded (human breast carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
8. Paraformaldehyde-fixed, paraffin embedded (mouse skin); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
9. Paraformaldehyde-fixed, paraffin embedded (mouse intestine); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
10. Paraformaldehyde-fixed, paraffin embedded (rat skin); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
11. Paraformaldehyde-fixed, paraffin embedded (rat intestine); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
12. Paraformaldehyde-fixed, paraffin embedded (human lung carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
13. Paraformaldehyde-fixed, paraffin embedded (human cervical carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
14. Paraformaldehyde-fixed, paraffin embedded (human Abdominal skin); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (TMAB-00159) at 1:100 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
15. Sample:
Lane 1: Mouse Cerebrum tissue lysates
Lane 2: Rat Cerebrum tissue lysates
Lane 3: Human HeLa cell lysates
Lane 4: Human Jurkat cell lysates
Lane 5: Human HL-60 cell lysates
Primary: Anti-ATF4 (TMAB-00159) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 38 kDa
Observed band size: 47 kDa

WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100; IF: 1:100-500; FCM: 0.2μg /test

Polyclonal

KLH conjugated synthetic peptide: human ATF4

Human

468