Sample:
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti- phospho-HMGCR (Ser872) (bs-4063R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 97 kD
Observed band size: 97 kD

Sample:
MCF-7(Human) Cell Lysate at 30 ug
Primary: Anti- phospho-HMGCR (Ser872) (bs-4063R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 97 kD
Observed band size: 97 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-HMGCR (Ser872)) Polyclonal Antibody, Unconjugated (bs-4063R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (phospho-HMGCR (Ser872)) Polyclonal Antibody, Unconjugated (bs-4063R) at 1:400 overnight at 4℃, followed by conjugation to the secondary antibody (labeled with HRP)and 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-HMGCR (Ser872)) Polyclonal Antibody, Unconjugated (bs-4063R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control: A431.
Primary Antibody (green line): Rabbit Anti-HMGCR antibody (bs-4063R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody: Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min 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.

Blank control (Black line): A431 (Black).
Primary Antibody (green line): Rabbit Anti-HMGCR antibody (bs-4063R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min 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.

phosphopeptide non phosphopeptide