α-Synuclein ER Stress Antibody Sampler Kit

Western blot analysis of DDIT3 on different lysates with Rabbit anti-DDIT3 antibody (ET1703-05) at 1/5,000 dilution.

Lane 1: SW480 cell lysate (20 µg/Lane)
Lane 2: HeLa cell lysate (20 µg/Lane)

Predicted band size: 19 kDa
Observed band size: 27 kDa

Exposure time: 3 minutes;

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 (ET1703-05) at 1/5,000 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.

Western blot analysis of Calnexin on different lysates with Rabbit anti-Calnexin antibody (ET1611-86) at 1/1,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-Calnexin KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 68 kDa
Observed band size: 90 kDa

Exposure time: 180 seconds; ECL: K1802;

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 (ET1611-86) at 1/1,000 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.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue with Rabbit anti-Calnexin antibody (ET1611-86) at 1/2,000 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 (ET1611-86) at 1/2,000 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.

Immunofluorescence analysis of frozen mouse cerebellum tissue with Rabbit anti-Alpha-Synuclein antibody (HA723035) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA723035, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Immunofluorescence analysis of frozen rat cerebellum tissue with Rabbit anti-Alpha-Synuclein antibody (HA723035) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA723035, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Western blot analysis of Alpha-Synuclein on different lysates with Rabbit anti-Alpha-Synuclein antibody (HA723035) at 1/2,000 dilution.

Lane 1: 293T transfected with empty control cell lysate
Lane 2: 293T transfected with Alpha-Synuclein cell lysate

Lysates/proteins at 15 µg/Lane.
Predicted band size: 14 kDa
Observed band size: 25 kDa
Exposure time: 3 minutes; 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 (HA723035) 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.

Western blot analysis of EIF2S1 on different lysates with Rabbit anti-EIF2S1 antibody (HA722112) at 1/2,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: HepG2 cell lysate
Lane 3: HeLa cell lysate
Lane 4: COS-1 cell lysate
Lane 5: A549 cell lysate
Lane 6: RAW264.7 cell lysate
Lane 7: C6 cell lysate
Lane 8: Mouse kidney tissue lysate
Lane 9: Mouse spleen tissue lysate
Lane 10: Rat kidney tissue lysate
Lane 11: Rat spleen tissue lysate

Lysates/proteins at 10 µg/Lane.
Predicted band size: 36 kDa
Observed band size: 36 kDa
Exposure time: 59 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 (HA722112) at 1/2,000 dilution and competitor's antibody at 1/2,000 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.

Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-GRP78 / BIP antibody (HA722202) at 1/1,000 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 (HA722202) at 1/1,000 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.

Western blot analysis of ATG12 on different lysates with Rabbit anti-ATG12 antibody (HA721504) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HCT 116 cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: SH-SY5Y cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 55/20 kDa

Exposure time: 3 minutes;

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 (HA721504) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

Flow cytometric analysis of HCT 116 cells labeling ATG12.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721504, 1ug/ml) (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).

All lanes: Western blot analysis of GBA with anti-GBA antibody [JM10-76] (ET1703-32) at 1:1,000 dilution.
Lane 1: Wild-type HEK293T whole cell lysate (20 µg).
Lane 2: GBA knockout HEK293T whole cell lysate (20 µg).

ET1703-32 was shown to specifically react with GBA in wild-type HEK293T cells. No band was observed when GBA knockout sample was tested. Wild-type and GBA knockout 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 (ET1703-32, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH , ET1601-4, 1/10,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.