Anti-HSP70 Polyclonal Antibody 2 is a Rabbit antibody targeting HSP70. Anti-HSP70 Polyclonal Antibody 2 can be used in WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Chicken,Cow,Rabbit,Sheep)

1. Paraformaldehyde-fixed, paraffin embedded (Mouse testis); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (HSP70) Polyclonal Antibody, Unconjugated (TMAB-00894) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
2. Paraformaldehyde-fixed, paraffin embedded (Rat testis); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (HSP70) Polyclonal Antibody, Unconjugated (TMAB-00894) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
3. Paraformaldehyde-fixed, paraffin embedded (Human breast carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (HSP70) Polyclonal Antibody, Unconjugated (TMAB-00894) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
4. Blank control: 293T.
Primary Antibody (green line): Rabbit Anti-HSP70 antibody (TMAB-00894)
Dilution: 2 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-FITC
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min 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.
5. Blank control: A549. Primary Antibody (green line): Rabbit Anti-HSP70 antibody (TMAB-00894)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF488
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. 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.
6. Blank control: A549. Primary Antibody (green line): Rabbit Anti-HSP70 antibody (TMAB-00894)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF488
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. 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.
7. Sample:
Lane 1: Testis (Mouse) Lysate at 40 μg
Lane 2: Epididymis (Mouse) Lysate at 40 μg
Lane 3: Seminal vesicle (Mouse) Lysate at 40 μg
Lane 4: Liver (Mouse) Lysate at 40 μg
Lane 5: NIH/3T3 (Mouse) Cell Lysate at 30 μg
Lane 6: Testis (Rat) Lysate at 40 μg
Lane 7: Spleen (Rat) Lysate at 40 μg
Lane 8: Hela (Human) Cell Lysate at 30 μg
Lane 9: Jurkat (Human) Cell Lysate at 30 μg
Lane 10: HepG2 (Human) Cell Lysate at 30 μg
Lane 11: A549 (Human) Cell Lysate at 30 μg
Lane 12: MCF-7 (Human) Cell Lysate at 30 μg
Primary:
Anti-HSP70 (TMAB-00894) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 70 kDa
Observed band size: 68 kDa
8. Tissue/cell: U-87MG cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (HSP70) polyclonal Antibody, Unconjugated (TMAB-00894) 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) was used to stain the cell nucleus.
9. 25 μg total protein per Lane of various lysates probed with HSP70 polyclonal antibody, unconjugated (TMAB-00894) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at RT for 60 min.

WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100; IF: 1:100-500; FCM: 1ug/Test

Polyclonal

KLH conjugated synthetic peptide: human HSP70

Human

Heat Shock Proteins,Tumor biomarkers