[IF=3.568] Hongyan Pei. et al. Network pharmacology and molecular docking analysis on the mechanism of Cordyceps militaris polysaccharide regulating immunity through TLR4/TNF-α pathwayss. J BIOCHEM MOL TOXIC. 2023 Apr;:e23345  WB ;  Mouse.  

PubMed:37050869

Sample:
Pancreas (Mouse) Lysate at 40 ug
Primary: Anti- phospho-beta Actin (Tyr53) (bs-12581R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD

Sample:
Lane 1: Hela (Human) Cell Lysate at 30 ug
Lane 2: MCF-7 (Human) Cell Lysate at 30 ug
Lane 3: Jurkat (Human) Cell Lysate at 30 ug
Primary:
Anti- phospho-beta Actin (Tyr53) (bs-12581R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD

Paraformaldehyde-fixed, paraffin embedded (rat stomach tissue); 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 (beta Actin (Tyr53)) Polyclonal Antibody, Unconjugated (bs-12581R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control: NIH/3T3.
Primary Antibody (green line): Rabbit Anti-phospho-beta Actin (Tyr53) antibody (bs-12581R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min 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.