Anti-HSP27 Polyclonal Antibody is a Rabbit antibody targeting HSP27. Anti-HSP27 Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

IgG

Human,Rat (predicted:Mouse,Dog,Pig,Cow)

1. Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01 M, pH 6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37℃ for 20 min;
Incubation: Anti-HSP-27 Polyclonal Antibody, Unconjugated (TMAB-07338) 1: 200, overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining
2. Tissue/cell: rat brain tissue; 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-HSP-27 Polyclonal Antibody, Unconjugated (TMAB-07338) 1: 200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated used at 1: 200 dilution for 40 minutes at 37°C. DAPI (5 μg/ml, blue) was used to stain the cell nuclei
3. Blank control (blue line): A431 cells (blue).
Primary Antibody (green line): Rabbit Anti-HSP27 antibody (TMAB-07338)
Dilution: 2 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% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. 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 (rat skeletal muscle); 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 (HSP27) Polyclonal Antibody, Unconjugated (TMAB-07338 ) at 1: 200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
5. Sample:
Lane 1: Hela (Human) Cell Lysate at 30 μg
Lane 2: MCF-7 (Human) Cell Lysate at 30 μg
Lane 3: A431 (Human) Cell Lysate at 30 μg
Lane 4: Huvec (Human) Cell Lysate at 30 μg
Lane 5: HepG2 (Human) Cell Lysate at 30 μg
Primary: Anti-HSP27 (TMAB-07338) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27-30 kD
Observed band size: 27 kD
6. Paraformaldehyde-fixed, paraffin embedded (rat stomach tissue); 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 (HSP27) Polyclonal Antibody, Unconjugated (TMAB-07338) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
7. Paraformaldehyde-fixed, paraffin embedded (Human breast cancer); 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 (HSP27) Polyclonal Antibody, Unconjugated (TMAB-07338) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
8. Paraformaldehyde-fixed, paraffin embedded (Rat uterus); 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 (HSP27) Polyclonal Antibody, Unconjugated (TMAB-07338) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 2μg/Test

Polyclonal

KLH conjugated synthetic peptide: human HSP27

Human

HSPB1

Heat shock protein beta-1

Involved in stress resistance and actin organization.