VALIDATION IMAGES
Sample:
SH-SY5Y (Human) Lysate at 40 ug
Primary:
Anti-beta-Actin (bs-0061R) at 1/2000~1/20000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Sample:Embryo Cerebrum (Mouse) Lysate at 40 ugDu145 (Human) Lysate at 40 ugSW480 (Human) Cell Lysate at 40 ugU87MG (Human) Lysate at 40 ugU251 (Human) Lysate at 40 ugA673 (Human) Lysate at 40 ugLovo (Human) Lysate at 40 ug293FT (Human) Lysate at 40 ugJEG-3 (Human) Lysate at 40 ugRSC96 (Rat) Cell Lysate at 40 ugMCF-7 (Human) Cell Lysate at 40 ugHepG2 (Human) Lysate at 40 ugA431 (Human) Lysate at 40 ugPrimary: Anti-beta-Actin (bs-0061R) at 1/2000 dilutionSecondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilutionPredicted band size: 42 kDObserved band size: 42 kD
Paraformaldehyde-fixed, paraffin embedded Human Breast Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Colon Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Small Intestine; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Rat Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Mouse Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Rat Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Mouse Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Rat Lung; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Mouse Lung; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with beta-Actin Polyclonal Antibody, Unconjugated (bs-0061R) at 1:200 overnight at 4°C, followed by conjugation to the SP Kit (Rabbit, SP-0023) and DAB (C-0010) staining.
Tissue/cell: Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (beta-Actin) polyclonal Antibody, Unconjugated (bs-0061R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG-FITC antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
MCF7 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (beta-Actin) polyclonal Antibody, Unconjugated (bs-0061R) 1:100, 90 minutes at 37°C; followed by a 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: NIH/3T3.
Primary Antibody (green line): Rabbit Anti-beta-Actin (Loading Control) antibody (bs-0061R)
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.