MLKL Recombinant Rabbit Monoclonal Antibody [SA40-04] - BSA and Azide free

☑ Relative expression (RE)

Western blot analysis of MLKL on different lysates with Rabbit anti-MLKL antibody (HA750017) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HUVEC cell lysate (20 µg/Lane)
Lane 2: HT-29 cell lysate (20 µg/Lane)
Lane 3: HeLa cell lysate (20 µg/Lane)
Lane 4: THP-1 cell lysate (20 µg/Lane)
Lane 5: U-937 cell lysate (20 µg/Lane)
Lane 6: K-562 cell lysate (low expression) (20 µg/Lane)
Lane 7: NIH/3T3 cell lysate (20 µg/Lane)

Predicted band size: 54 kDa
Observed band size: 54 kDa

Exposure time: 1 minute 2 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750017) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of MLKL on different lysates with Rabbit anti-MLKL antibody (HA750017) at 1/5,000 dilution.

Lane 1: Hela-si NT cell lysate (10 µg/Lane)
Lane 2: Hela-si MLKL cell lysate (10 µg/Lane)

Predicted band size: 54 kDa
Observed band size: 54 kDa

Exposure time: 3 minutes 20 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

ET1601-25 was shown to specifically react with MLKL in Hela-si NT cells. No band was observed when Hela-si MLKL samples were tested. Hela-si NT and Hela-si MLKL samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1601-25, 1/5,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at 4 ℃ overnight. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of HeLa cells labeling MLKL with Rabbit anti-MLKL antibody (HA750017) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-MLKL antibody (HA750017) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of HT-29 cells labeling MLKL with Rabbit anti-MLKL antibody (HA750017) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-MLKL antibody (HA750017) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of HT-29 cells labeling MLKL.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750017, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Application: Immunohistochemistry (IHC-P)

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

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

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: HA750017, 1/2,000, 1 hour at room temperature.
Secondary antibody: HA1119, 20 minutes at room temperature.

Application: Immunofluorescence (IF-tissue)

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

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

Wash buffer: 1× PBS
Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes.
Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
Primary antibody: HA750017, 1/1,000, overnight at 4℃.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 1.5 hours at room temperature.