Sample: Lung (Mouse) Lysate at 40 ug
Primary: Anti-phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305) (SL5182R) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (SL0295G-HRP) at 1/5000 dilution
Predicted band size: 53 kD
Observed band size: 53 kD
Sample:
Hela(Human) Cell Lysate at 30 ug
HepG2(Human) Cell Lysate at 30 ug
Primary: Anti-phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305) (SL5182R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 62/53 kD
Observed band size: 62/53 kD
Sample:
NIH/3T3(Mouse) Cell Lysate at 30 ug
Adrenal gland(Mouse) Lysate at 40 ug
MCF-7(Human) Cell Lysate at 30 ug
A549(Human) Cell Lysate at 30 ug
Primary: Anti-phospho-AKT1+AKT2+AKT3 (Thr308+Thr309+Thr305) (SL5182R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 60 kD
Observed band size: 60 kD
Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305)) Polyclonal Antibody, Unconjugated (SL5182R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305)) Polyclonal Antibody, Unconjugated (SL5182R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Antibody, Unconjugated(SL5182R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: A549 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305)) polyclonal Antibody, Unconjugated (SL5182R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control(blue):EC9706 (fixed with 2% paraformaldehyde for 10 min at 37℃).
Primary Antibody:Rabbit Anti-phospho-AKT1 + AKT2 + AKT3 (Thr308+Thr309+Thr305) antibody (SL5182R,Green); Dilution: 1μg 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-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
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