MLKL Recombinant Rabbit Monoclonal Antibody [SA40-04]
cat.: HA750017
| Product Type: | Recombinant Rabbit monoclonal IgG, primary antibodies |
|---|---|
| Species reactivity: | Human, Mouse |
| Applications: | WB, IHC-P, IF-Cell, FC, IF-Tissue |
| Clonality: | Monoclonal |
| Clone number: | SA40-04 |
| Form: | Liquid |
| Storage condition: | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage buffer: | 1*PBS (pH7.4). |
| Concentration: | 1ug/ul |
| Purification: | Protein A affinity purified. |
| Molecular weight: | Predicted band size: 54 kDa |
| Isotype: | IgG |
| Immunogen: | Recombinant protein within Human MLKL aa 333-471 / 471. |
| Positive control: | HUVEC cell lysate, HT-29 cell lysate, HeLa cell lysate, THP-1 cell lysate, U-937 cell lysate, K-562 cell lysate, NIH/3T3 cell lysate, HeLa, HT-29, human tonsil tissue. |
| Subcellular location: | Cytoplasm, Cell membrane, Nucleus. |
| Recommended Dilutions:
WB IHC-P IF-Cell FC IF-Tissue |
1:5,000-1:10,000 1:500-1:2000 1:100 1:1,000 1:500-1:1,000 |
| Uniprot #: | SwissProt: Q8NB16 Human | Q9D2Y4 Mouse |
| Alternative names: | 9130019I15Rik FLJ34389 hMLKL Mixed lineage kinase domain like Mixed lineage kinase domain like protein Mixed lineage kinase domain-like protein Mlkl MLKL_HUMAN |
Images
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Fig1:
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. |
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Fig2:
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. |
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Fig3:
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. |
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Fig4:
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. |
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Fig5:
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). |
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Fig6:
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. |
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Fig7:
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. |
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.