Antibody Type: Recombinant Rabbit Monoclonal

Application: ELISA, WB, IHC, IF, FCM, IP

Reactivity: Human

Rabbit IgG

3H943

Human

1. Western Blot
-Positive WB detected in: Hela whole cell lysate, PC3 whole cell lysate, 293T whole cell lysate, LO2 whole cell lysate, HT-29 whole cell lysate, K562 whole cell lysate
-All lanes: Hsp27 antibody at 0.62μg/ml
-Secondary: Goat polyclonal to rabbit IgG at 1/50000 dilution
-Predicted band size: 23 KDa
-Observed band size: 27 KDa
2. IHC image of TMAH-00580 diluted at 1:61.9 and staining in paraffin-embedded human pancreatic tissue performed on a Leica BondTM system. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated SP system.
3. IHC image of TMAH-00580 diluted at 1:61.9 and staining in paraffin-embedded human cervical cancer performed on a Leica BondTM system. After dewaxing and hydration, antigen retrieval was mediated by high pressure in a citrate buffer (pH 6.0). Section was blocked with 10% normal goat serum 30min at RT. Then primary antibody (1% BSA) was incubated at 4°C overnight. The primary is detected by a biotinylated secondary antibody and visualized using an HRP conjugated SP system.
4. Immunofluorescence staining of MCF-7 cells with TMAH-00580 at 1:20, counter-stained with DAPI. The cells were fixed in 4% formaldehyde, permeabilized using 0.2% Triton X-100 and blocked in 10% normal Goat Serum. The cells were then incubated with the antibody overnight at 4°C. The secondary antibody was Alexa Fluor 488-congugated AffiniPure Goat Anti-Rabbit IgG (H+L).
5. Immunoprecipitating Hsp27 in Hela whole cell lysate
-Lane 1: Rabbit control IgG instead of TMAH-00580 in Hela whole cell lysate.
For western blotting, a HRP-conjugated Protein G antibody was used as the secondary antibody (1/2000)
-Lane 2: TMAH-00580 (3μg) + Hela whole cell lysate (500μg)
-Lane 3: Hela whole cell lysate (20μg)
6. Overlay histogram showing Hela cells stained with TMAH-00580 (red line) at 1:50. The cells were fixed with 70% Ethylalcohol (18h) and then permeabilized with 0.3% Triton X-100 for 2 min. The cells were then incubated in 1x PBS /10% normal goat serum to block non-specific protein-protein interactions followed by primary antibody for 1 h at 4°C. The secondary antibody used was FITC goat anti-rabbit IgG (H+L) at 1/200 dilution for 1 h at 4°C. Control antibody (green line) was used under the same conditions. Acquisition of >10,000 events was performed.

ELISA, WB, IHC, IF, FCM, IP

WB:1:500-1:5000; IHC:1:50-1:200; IF:1:20-1:200; IP:1:200-1:1000.

Monoclonal

Unconjugated

A synthetic peptide: Human HSPB1

Human

3315

Signal Transduction