Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon Polyclonal Antibody is a Rabbit antibody targeting 14-3-3 Alpha+Beta+Gamma+Delta+Epsilon. Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Sheep,Fruit Fly,Yeast)

1. Tissue/cell: human rectal carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01 M, pH 6.0), Boiling bathing for 15 min; Blocking buffer (normal goat serum) at 37℃ for 20 min;
Incubation: Anti-14-3-3 Polyclonal Antibody, Unconjugated (TMAB-02075) 1: 200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, PE conjugated used at 1: 200 dilution for 40 minutes at 37°C. DAPI (5 μg/ml, blue) was used to stain the cell nuclei
2. Sample: Brain Cell Lysate at 40 μg
Primary: Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon (TMAB-02075) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27 kD
Observed band size: 27 kD
3. Blank control (blue line): A549 (blue).
Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon antibody (TMAB-02075)
Dilution: 1 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℃) 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. Acquisition of 20,000 events was performed.
4. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); 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 (14-3-3 Alpha+Beta+Gamma+Delta+Epsilon) Polyclonal Antibody, Unconjugated (TMAB-02075) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.
5. Sample:
Cerebrum (Mouse) Lysate at 40 μg
Primary: Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon (TMAB-02075) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27 kD
Observed band size: 27 kD
6. Paraformaldehyde-fixed, paraffin embedded (mouse brain); 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 (14-3-3 Alpha+Beta+Gamma+Delta+Epsilon) Polyclonal Antibody, Unconjugated at 1: 200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
7. Paraformaldehyde-fixed, paraffin embedded (rat brain); 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 (14-3-3 Alpha+Beta+Gamma+Delta+Epsilon) Polyclonal Antibody, Unconjugated at 1: 200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
8. Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 μg
Lane 2: Uterus (Mouse) Lysate at 40 μg
Lane 3: Testis (Mouse) Lysate at 40 μg
Lane 4: Liver (Mouse) Lysate at 40 μg
Lane 5: Lung (Mouse) Lysate at 40 μg
Lane 6: Cerebrum (Rat) Lysate at 40 μg
Lane 7: Uterus (Rat) Lysate at 40 μg
Lane 8: Testis (Rat) Lysate at 40 μg
Lane 9: Liver (Rat) Lysate at 40 μg
Lane 10: Lung (Rat) Lysate at 40 μg
Lane 11: NIH/3T3 (Mouse) Cell Lysate at 30 μg
Lane 12: Siha (Human) Cell Lysate at 30 μg
Lane 13: U251 (Human) Cell Lysate at 30 μg
Primary: Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon (TMAB-02075) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 28 kD
Observed band size: 28 kD
9. Blank control: A431.
Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon antibody (TMAB-02075)
Dilution: 1 μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: 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 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 20,000 events was performed.

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

Polyclonal

KLH conjugated synthetic peptide: human 14-3-3

Human

YWHAB

14-3-3 protein beta/alpha

Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner.