alpha Actinin 4 Recombinant Rabbit Monoclonal Antibody [JU20-23]
cat.: ET1706-05
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IP, IHC-P, FC
Clonality: Monoclonal
Clone number: JU20-23
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 105 kDa
Isotype: IgG
Immunogen: Synthetic peptide within Human alpha Actinin 4 aa 1-50 / 911.
Positive control: A549 cell lysate, HeLa cell lysate, NIH/3T3 cell lysate, Mouse lung tissue lysate, Rat liver tissue lysate, Rat lung tissue lysate, A549, NIH/3T3, mouse liver tissue, rat liver tissue.
Subcellular location: Cell junction. Cytoplasm. Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IP

1:2,000
1:100-1:500
1:200
1:1,000
1-2μg/sample
Uniprot #: SwissProt: O43707 Human | P57780 Mouse | Q9QXQ0 Rat
Alternative names: actinin 4 Actinin alpha 4 actinin4 ACTN 4 ACTN4 ACTN4_HUMAN Alpha-actinin-4 DKFZp686K23158 F actin cross linking protein F-actin cross-linking protein Focal segmental glomerulosclerosis 1 FSGS 1 FSGS FSGS1 Non muscle alpha actinin 4 Non-muscle alpha-actinin 4
Images
ET1706-05_1.jpg Fig1: Western blot analysis of alpha Actinin 4 on different lysates with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/2,000 dilution.

Lane 1: A549-si NT cell lysate
Lane 2: A549-si alpha Actinin 4 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 105 kDa
Observed band size: 100 kDa

Exposure time: 30 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 (ET1706-05) at 1/2,000 dilution was 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.
ET1706-05_2.jpg Fig2: Western blot analysis of alpha Actinin 4 on different lysates with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/2,000 dilution.

Lane 1: A549 cell lysate
Lane 2: HeLa cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: Mouse lung tissue lysate
Lane 5: Rat liver tissue lysate
Lane 6: Rat lung tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 105 kDa
Observed band size: 100 kDa

Exposure time: 20 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 (ET1706-05) at 1/2,000 dilution was 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.
ET1706-05_3.jpg Fig3: Immunocytochemistry analysis of A549 cells labeling alpha Actinin 4 with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-alpha Actinin 4 antibody (ET1706-05) 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 (HA601187, 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.
ET1706-05_4.jpg Fig4: Immunocytochemistry analysis of NIH/3T3 cells labeling alpha Actinin 4 with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-alpha Actinin 4 antibody (ET1706-05) 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 (HA601187, 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.
ET1706-05_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1706-05) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1706-05_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded rat liver tissue with Rabbit anti-alpha Actinin 4 antibody (ET1706-05) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1706-05) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1706-05_7.jpg Fig7: Flow cytometric analysis of A549 cells labeling alpha Actinin 4.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1706-05, 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).
ET1706-05_8.jpg Fig8: Flow cytometric analysis of NIH/3T3 cells labeling alpha Actinin 4.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1706-05, 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).
ET1706-05_9.jpg Fig9: alpha Actinin 4 was immunoprecipitated from 0.2 mg A549 cell lysate with ET1706-05 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1706-05 at 1/1,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A549 cell lysate (input)
Lane 2: ET1706-05 IP in A549 cell lysate
Lane 3: Rabbit IgG instead of ET1706-05 in A549 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 3 seconds; ECL: K1801
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.