Phospho-Beta Catenin (S675) Recombinant Rabbit Monoclonal Antibody [PSH25-60] - BSA and Azide free

☑ Cell treatment (CT)

Western blot analysis of Phospho-Beta Catenin (S675) on different lysates with Rabbit anti-Phospho-Beta Catenin (S675) antibody (HA751979) at 1/5,000 dilution.

Lane 1: Mouse brain tissue lysate
Lane 2: Mouse liver tissue lysate
Lane 3: Mouse brain tissue lysate, the membrane treated with λpp for 1 hour
Lane 4: Rat brain tissue lysate
Lane 5: Rat liver tissue lysate

Lysates/proteins at 20 µg/Lane.
Exposure time: Lane 1-3: 1 minute; Lane 4-5: 2 minutes; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA751979, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 °C
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 85 kDa
Observed band size: 85 kDa

Application: Immunofluorescence (IHC-Fr)

Species: Mouse
Tissue: Colon
Sample: Frozen section

Antigen retrieval: Not required

Wash buffer: 1× TBST
Blocking: 10% normal goat serum + 1% Triton X-100 + 0.3 M Glycine in TBST, 30 minutes at room temperature.

Primary antibody: HA751979, 1/1,000, overnight at 4°C.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Human
Tissue: Colon
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95°C.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA751979, 1/4,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

☑ Cell treatment (CT)

Application: Immunohistochemistry (IHC-P)

Species: Mouse
Tissue: Colon
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95°C.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA751979, 1/4,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunohistochemistry (IHC-P)

Species: Rat
Tissue: Colon
Sample: Paraffin-embedded section

Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95°C.

Wash buffer: 1× TBST
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA751979, 1/4,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Immunoprecipitation (IP)

Phospho-Beta Catenin (S675) was immunoprecipitated in 0.2 mg mouse brain tissue lysate with HA751979 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA751979 at 1/5,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: Mouse brain tissue lysate (input)
Lane 2: HA751979 IP in mouse brain tissue lysate
Lane 3: Rabbit IgG instead of HA751979 in mouse brain tissue lysate

Exposure time: 25 seconds
Blocking: 5% NFDM/TBST, 1 hour at room temperature

Primary dilution: HA751979, 1/5,000 in primary antibody dilution buffer (K1803), 2 hours at room temperature

Predicted band size: 85 kDa
Observed band size: 85 kDa